The effect of lipids from mycobacterium bovis on bovine ...etheses.bham.ac.uk/5663/1/Pirson15PhD.pdf · Pirson et al., (2012) “Differential effects of Mycobacterium bovis - derived

The Effects of Lipids from Mycobacterium

bovis on Bovine Innate and Acquired

Immune Responses

By

Christopher Pirson

A thesis submitted to the University of Birmingham

for the Degree of Doctor of Philosophy

Bovine Tuberculosis Research Team

Animal Health & Veterinary Laboratories Agency

&

School of Biosciences

University of Birmingham

September 2014

University of Birmingham Research Archive

e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder.

Declaration

The work presented in this thesis was carried out in the Bovine TB Research Team at the

Animal Health and Veterinary Laboratories Agency KT15 3NB, during the period

September 2009 to September 2014. The work in this thesis is original except where

acknowledged by references.

No portion of the work is being, or has been submitted for a degree, diploma or any other

qualification at any other University.

ii

Published Work Declaration

This thesis contains work which has been accepted to and / or published in peer -

reviewed journals. Any publications arising from the work presented in this thesis were

written and prepared by Christopher Pirson who is listed as the publications primary

author and the contributions of other authors to the text in this thesis are not substantial.

Thesis content which is similar to that contained in associated peer - reviewed

publications is listed below:

Pirson et al., (2012) “Differential effects of Mycobacterium bovis - derived polar and apolar lipid fractions on bovine innate immune cells” Veterinary Research 43:54 • Chapter One: pages 6, 10 and 31 • Chapter Two sections:

o Preparation of Bacterial Isolates for Lipid Extraction o Extraction of Crude Free Mycobacterial Lipids o Analysis of Lipid Fractions by 2D Thin Layer Chromatography o Preparation of Lipid Antigen Suspensions o Uninfected Cattle o Isolation of Bovine PBMC from Whole Blood o Isolation of CD14+ Monocytes from Bovine PBMC o Generation of Bovine Cultured Monocytes and MDDC o Multiplex Measurement of Cytokine Production o Innate Cell Labelling & Analysis by Flow Cytometry o Mixed Lymphocyte Reaction

• Chapter Three, Discussion: page 70 • Chapter Four sections:

o Results o Discussion

iii

Pirson et al., (2015) “Highly purified mycobacterial phosphatidylinositol mannosides drive cell mediated responses and activate NKT cells in cattle” Clinical and Vaccine Immunology 22:2 • Chapter Two sections:

o Preparation of Lipid Antigen Suspensions o M. bovis Infected Cattle o Isolation of Bovine PBMC from Whole Blood o Measurement of IFNγ by BovigamTM ELISA o Lymphocyte Transformation Assay o Lymphocyte Labelling & Analysis by Flow Cytometry

• Chapter Six: pages 130, 131 and 137 - 138

Signed:

C. Pirson

H. M. Vordermeier

iv

Abstract

The interaction between the host and the pathogen is critical in defining the

outcome of an infection. For cattle with bovine tuberculosis (BTB), this interaction is

likely to occur between antigen presenting cells in the lung and the lipid rich surface of

the causative agent Mycobacterium bovis (M. bovis). It is well documented that lipid

molecules from mycobacteria are capable of modulating immune responses however

previous work has made use of model animal systems or lipids from avirulent bacteria.

The aim of this study was to extract lipids from virulent M. bovis and assess any

immunomodulatory ability of these molecules in cattle with a view to aiding in the

development of control measures for BTB.

To this end, lipids were extracted from M. bovis AF 2122/97 and AN5 and the fractions

characterised. Upon thin layer chromatography analysis, polar and apolar fractions from

both bacterial strains were found to be broadly similar in their lipid constitution although

quantitative differences were noted. Lipopeptide was also identified in both polar

fractions. Stimulation of bovine antigen presenting cells with the lipid fractions showed

polar lipids mediated increases in IL - 10 and IL - 12 production and reductions in cell

surface expression of MHCII and CD1b. Further investigation of the polar lipid fraction

was performed by subfractionation but no individual lipid could be found responsible for

the responses of antigen presenting cells to these subfractions.

The ability of the polar and apolar lipid fractions to be recognised by cells of the adaptive

immune system was assessed and the polar fraction was found to drive production of

IFNγ and strong proliferation of bovine lymphocytes. The role of lipopeptide in the polar

fraction was evaluated by enzymatic degradation with Proteinase K and blockade of

either MHCII or CD1. While lipopeptide was found to play a role in the generation of

lymphocyte responses, these treatments did not abrogate the effects completely

suggesting a lipid mediated component as well. Screening of highly purified individual

lipid molecules led to the selection of one molecule (AcPIM6), which was found to be

capable of driving antigen specific proliferation of NKT cells.

v

Dedication

For the cows…

vi

Acknowledgements

First and foremost, I would like to thank my supervisor at AHVLA, Prof Martin

Vordermeier. Martin provided me with both the inspiration and the self - belief that enabled me

to undertake this PhD and without his constant support I would not be where I am today. Rarely

will you meet a man of such wisdom and international repute who gives their time and

knowledge so freely. I also want to recognise the indefatigable encouragement from Dr Gareth

Jones, who may not have signed up to supervise a PhD but took on the responsibility with good

grace and humour. His scientific knowledge, rigour and attention to detail are aspirational and he

helped hammer both the experiments and this thesis into shape.

I must also thank Prof Del Besra and his staff at the University, particularly Dr Sid Gurcha who

provided much support during my time in Birmingham and without whom the completion of this

project would not have been possible.

I would also like to express my gratitude to my professional collaborators; particularly Prof Otto

Holst from the Research Centre Borstel for supplying both reagents and good advice in equal

measure, and Dr Arun Mishra (who I met in Birmingham but is now at NIMR) who provided me

with many of his methods for lipid purification. I would be remiss if I didn’t thank Dr Max Bastian

who lent his technical assistance and his expertise as well as the methods for Proteinase K

digestion of the lipid fractions. My thanks also go to Prof Mark Chambers and the former

Tuberculin Production Unit at AHVLA Weybridge for the pellicle of AN5 which enabled this much

of the work in this project.

vii

I am indebted to my colleagues, past and present, at AHVLA Weybridge for their support and

(occasionally unenthusiastic) help. To Adam, Bernardo, Phil, Tom, Laura, Ilaria, Gareth, Carmen,

Jim, Shelley, Gilly, Stefan, Paul W, Mick, Roland, Paul A, Sonya, Daryan, Emma, Peter, Karen,

Krista, Richard, Elihu, Holly, Margot and Penny I offer my heartfelt thanks. Much has happened in

the past few years and I wouldn’t have made it through without their friendship, support and

(mostly) friendly abuse. A special thanks is reserved for Dr Paul Wheeler whose peace and quiet I

shattered by moving into his lab to perform my lipid analysis work and whose brain I picked on a

regular basis.

Over the past few years I have garnered much support via Twitter. The #PhDchat hashtag has

proven a valuable resource and several users have provided me with advice, support and silly

things to laugh at. @LunaLevitt, @PayalYokota, @curexcomplex, @jamimmunology,

@psyoureanidiot, @_MaddieHoward, @CS_Diamond, @Carly0308 and @Gemgemloulou have all

been more helpful than they probably realise.

It really only remains for me to thank my future wife Julia and my family; their support and belief

has meant a great deal to me and I hope to return the favour one day.

viii

Table of Contents

Declaration ......................................................................................................................................... ii

Published Work Declaration .............................................................................................................. iii

Abstract .............................................................................................................................................. v

Dedication ......................................................................................................................................... vi

Acknowledgements .......................................................................................................................... vii

Table of Contents .............................................................................................................................. ix

List of Figures ................................................................................................................................... xii

List of Tables ..................................................................................................................................... xv

Publications Associated with this Thesis .......................................................................................... xvi

Posters Associated with this Thesis ................................................................................................. xvi

List of Abbreviations ........................................................................................................................ xvii

Introduction ....................................................................................................................................... 1

The Mycobacteria ........................................................................................................................... 1

The Mycobacterium tuberculosis Complex .................................................................................... 2

Mycobacterium tuberculosis ...................................................................................................... 3

Mycobacterium bovis ................................................................................................................. 5

Control of BTB In Great Britain ....................................................................................................... 7

New Strategies for BTB Control.................................................................................................... 11

Mycobacterial Lipids .................................................................................................................... 17

Immune Recognition of Lipids .................................................................................................. 22

Lipid Modulation of Innate Immune Responses ...................................................................... 23

Lipid Recognition by Adaptive Immunity ................................................................................. 26

The Aims of this Study .................................................................................................................. 30

Materials & Methods ....................................................................................................................... 34

Preparation of Bacterial Isolates for Lipid Extraction .................................................................. 34

Extraction of Crude Free Mycobacterial Lipid .............................................................................. 35

Analysis of Lipid Fractions by 2D Thin Layer Chromatography .................................................... 37

Thin Layer Chromatography Densitometrical Analysis ................................................................ 39

Preparation of Lipid Antigen Suspensions ................................................................................... 39

Lipid Purification by 1D Thin Layer Chromatography .................................................................. 40

Uninfected Cattle ......................................................................................................................... 42

M. bovis Infected Cattle ............................................................................................................... 42

ix

Isolation of Bovine PBMC from Whole Blood .............................................................................. 43

Isolation of CD14+ Monocytes from Bovine PBMC ...................................................................... 43

Generation of Bovine Cultured Monocytes and MDDC ............................................................... 43

Measurement of IFNγ by BovigamTM ELISA .................................................................................. 44

Multiplex Measurement of Cytokine Production ........................................................................ 44

Innate Cell Labelling & Analysis by Flow Cytometry .................................................................... 45

Mixed Lymphocyte Reaction ........................................................................................................ 47

Lymphocyte Transformation Assay .............................................................................................. 48

Lymphocyte Labelling & Analysis by Flow Cytometry .................................................................. 49

Monoclonal Antibody Blocking of MHCII and CD1 ....................................................................... 50

Removal of Lipopeptide by Proteinase Treatment ...................................................................... 51

Data & Statistical Analysis ............................................................................................................ 51

Preparation & Characterisation of Crude Lipid Extracts .................................................................. 52

Background .................................................................................................................................. 52

Results .......................................................................................................................................... 56

Extraction & Analysis of Lipids from M. bovis AF 2122/97 ...................................................... 56

Extraction & Analysis of Lipids from M. bovis AN5 .................................................................. 60

Abundance Analysis of the Crude Lipid Fractions .................................................................... 64

Discussion ..................................................................................................................................... 70

Chapter Summary ........................................................................................................................ 76

Effects of Crude Lipids on Bovine Innate Immune Cells ................................................................... 77

Background .................................................................................................................................. 77

Results .......................................................................................................................................... 80

Characterisation of Cultured Monocytes and Monocyte Derived DC ...................................... 80

Cytokine Responses to Crude Mycobacterial Lipids ................................................................ 85

Phenotypic Responses to Crude Mycobacterial Lipids ............................................................ 88

Consequence of MDDC Exposure to M. bovis - Derived Lipids ................................................ 90

Discussion ..................................................................................................................................... 92

Chapter Summary ........................................................................................................................ 97

Effects of Lipid Subfractions on Bovine Innate Immune Cells .......................................................... 98

Background .................................................................................................................................. 98

Results ........................................................................................................................................ 101

The Effect of Crude Polar Lipids from M. bovis AN5 .............................................................. 101

Subfractionation of the Crude Polar Lipids from M. bovis AN5 ............................................. 102

x

Cytokine & Phenotypic Responses to Lipid Subfractions ....................................................... 107

Assessment of Lipopeptide presence in Lipid Subfractions ................................................... 112

Discussion ................................................................................................................................... 115

Chapter Summary ...................................................................................................................... 122

Effect of Lipids on Bovine Acquired Cell - Mediated Immunity ..................................................... 123

Background ................................................................................................................................ 123

Results ........................................................................................................................................ 126

Lymphocyte Responses to Crude Mycobacterial Lipids ......................................................... 126

Lipopeptide Activity in the Crude Polar Fraction ................................................................... 128

Adaptive Immune Responses to Purified PIM Molecules ...................................................... 131

Phenotyping of AcPIM6 Responsive Cells by Flow Cytometry................................................ 132

Discussion ................................................................................................................................... 135

Chapter Summary ...................................................................................................................... 140

Concluding Remarks ....................................................................................................................... 141

Bibliography ................................................................................................................................... 145

Appendix ........................................................................................................................................ 217

Publications Associated with this Thesis .................................................................................... 217

xi

List of Figures

Figure 1:1 - BTB testing areas shown for the year 2014 ....................................................... 8

Figure 1:2 - Incidence of BTB in GB since 1996 ................................................................... 11

Figure 1:3 - Model of the mycobacterial cell wall. .............................................................. 20

Figure 1:4 - Structures of some common outer envelope lipids found in virulent

mycobacteria ....................................................................................................................... 21

Figure 2:1 - Schematic representation of the extraction of free mycobacterial lipids. ...... 37

Figure 2:2 - Representative 1D TLC of crude free polar mycobacterial lipids .................... 41

Figure 2:3 - Example of gating strategy for analysis of monocytes, MDM and MDDC ....... 47

Figure 2:4 - Example of gating strategy for analysis of proliferative cells .......................... 50

Figure 3:1 - 2D TLC analysis of crude, free lipids extracted from M. bovis AF 2122/97 and

stained with MPA ................................................................................................................ 57

Figure 3:2 - 2D TLC analysis of crude, free lipids extracted from M. bovis AF 2122/97 and

stained with ninhydrin ......................................................................................................... 59

Figure 3:3 - 2D TLC analysis of crude, free lipids extracted from M. bovis AN5 and stained

with MPA ............................................................................................................................. 61

Figure 3:4 - 2D TLC analysis of crude, free lipids extracted from M. bovis AN5 and stained

with ninhydrin ..................................................................................................................... 63

Figure 3:5 - False coloured densitometry analysis of lipids extracted from M. bovis AF

2122/97 and analysed by 2D TLC ........................................................................................ 65

Figure 3:6 - False coloured densitometry analysis of lipids extracted from M. bovis AN5

and analysed by 2D TLC ....................................................................................................... 67

xii

Figure 4:1 - CD14+ cells after culture for 3 days in the presence of either GM - CSF or GM -

CSF and IL - 4. ....................................................................................................................... 81

Figure 4:2 - Phenotype of fresh CD14+ monocytes, cultured monocytes (CM) and cultured

DC (MDDC) ........................................................................................................................... 83

Figure 4:3 - Effect of stimulation with the polar and apolar lipid fractions on cytokine

production by bovine innate immune cells. ........................................................................ 86

Figure 4:4 - Effect of stimulation with the polar and apolar lipid fractions on phenotype of

bovine innate immune cells. ............................................................................................... 89

Figure 4:5 - Proliferative responses of PBMC stimulated with polar lipid treated allotypic

cultured monocytes and MDDC .......................................................................................... 91

Figure 5:1 - Effect of polar lipids from M. bovis AF 2122/97 and AN5 on phenotype of

bovine MDDC. .................................................................................................................... 102

Figure 5:2 - Subfractionation by glass column chromatography. ..................................... 103

Figure 5:3 - Subfractionation by solid phase extraction chromatography. ...................... 104

Figure 5:4 - One dimensional TLC of the polar lipid subfractions. .................................... 105

Figure 5:5 - 2D TLC analysis of the 6 polar lipid subfractions stained with MPA .............. 106

Figure 5:6 - Effect of polar lipids from M. bovis AN5 on cytokine production by bovine

MDDC ................................................................................................................................. 108

Figure 5:7 - Effect of lipid subfractions from M. bovis AN5 on phenotype of bovine MDDC

........................................................................................................................................... 109

Figure 5:8 - Effect of serially diluted polar lipids from M. bovis AN5 on IL - 10 production

by bovine MDDC ................................................................................................................ 110

Figure 5:9 - Effect of serially diluted polar lipids from M. bovis AN5 on IL - 12 production

by bovine MDDC ................................................................................................................ 111

xiii

Figure 5:10 - Effect of serially diluted polar lipids from M. bovis AN5 on MHCII expression

by bovine MDDC ................................................................................................................ 112

Figure 5:11 - 2D TLC analysis of the 6 polar lipid subfractions stained with ninhydrin .... 113

Figure 6:1 - Effect of stimulation with the AF 2122/97 polar and apolar lipid fractions on

bovine PBMC. .................................................................................................................... 126

Figure 6:2 - Effect of stimulation with the AF 2122/97 polar and AN5 polar lipid fractions

on bovine PBMC. ............................................................................................................... 127

Figure 6:3 - Effect of blocking CD1 and MHCII on AN5 polar fraction driven cell - mediated

responses. .......................................................................................................................... 129

Figure 6:4 - Effect of Proteinase K treatment on proliferative ability of the AN5 polar lipid

fraction. ............................................................................................................................. 130

Figure 6:5 - Effect of stimulation with purified PIMs on bovine PBMC. ........................... 132

Figure 6:6 - Assessment of proliferating cell phenotype by flow cytometry .................... 133

xiv

List of Tables

Table 2:1 - Solvent systems for TLC analysis of mycobacterial lipids (adapted from Dobson

et al.(252)) .............................................................................................................................. 38

Table 3:1 - Densitometry of the apolar lipid fractions. ....................................................... 68

Table 3:2 - Densitometry of the polar lipid fractions. ......................................................... 68

xv

Publications Associated with this Thesis

C. Pirson, G. J. Jones, S. Steinbach, G. S. Besra & H. M. Vordermeier, (2012). “Differential

effects of Mycobacterium bovis - derived polar and apolar lipid fractions on bovine

innate immune cells” Veterinary Research 43 : 54

C. Pirson, R. Engel, G. J. Jones, T. Holder, O. Holst & H. M. Vordermeier. “Highly purified

mycobacterial phosphatidylinositol mannosides drive cell mediated responses and

activate NKT cells in cattle” Clinical and Vaccine Immunology 22 : 2

Posters Associated with this Thesis

VI International M. bovis Conference

Differential effects of Mycobacterium bovis - derived polar and apolar lipid fractions on

bovine innate immune cells

xvi

List of Abbreviations

BTB Bovine Tuberculosis

CIITA Class II Major Histocompatibility Complex Transactivator

CD Cluster of Differentiation

CFP - 10 Culture Filtrate Protein - 10 kDa

CM Cultured Monocyte

CR Complement Receptor

DAT Diacyl Trehalose

DC Dendritic Cell

DC - SIGN Dendritic Cell - Specific Intercellular Adhesion Molecule - 3 Grabbing Nonintegrin

DPG Diphosphatidyl Glycerol

DPH 1, 6 - diphenyl - 1, 3, 5 - hexatriene

ESAT - 6 Early Secreted Antigenic Target 6 kDa

GB Great Britain

GM - CSF Granulocyte - Macrophage Colony Stimulating Factor

IFN Interferon

IL Interleukin

kDa Kilodalton

LAM Lipoarabinomannan

ManLAM Mannose Capped Lipoarabinomannan

MAPK Mitogen - Activated Protein Kinase

MDDC Monocyte Derived Dendritic Cell

MFI Median Fluorescence Intensity

MHC Major Histocompatibility Complex

MIP Macrophage Inflammatory Protein

MMG Monomycolyl Glycerol

MPA Molybdophosphoric Acid

MQ Menaquinone

MR Mannose Receptor

NFκB Nuclear Factor kappa - Light Chain Enhancer of Activated B Cells

NIRViD Near Infra - Red Viability Dye

NLR Nucleotide Oligomerisation Domain - Like Receptor

NOD Nucleotide Oligomerisation Domain

PBMC Peripheral Blood Mononuclear Cells

PBS Phosphate Buffered Saline

PDIM Phthiocerol Dimycocerosate

xvii

PE Phosphatidyl Ethanolamine

PGL Phenolic Glycolipid

PI Phosphatidylinositol

PIM Phosphatidylinositol Mannoside

PPD - A Purified Protein Derivative - Avian

PPD - B Purified Protein Derivative - Bovine

PRR Patern Recognition Receptor

PWM Pokeweed Mitogen

R - PE R - Phycoerythrin RLU Relative Light Units

RPMI Roswell Park Memorial Institute

SGL Sulphoglycolipid

SL Sulpholipid

TAG Triacyl Glycerol

TCR T Cell Receptor

TDB Trehalose Dibehenate

TDM Trehalose Dimycolate

Th T Helper

TLC The Layer Chromatography

TLR Toll - Like Receptor

TMM Trehalose Monomycolate

TNF Tumour Necrosis Factor

ViViD Violet Viability Dye

xviii

Chapter One Introduction

Chapter One

Introduction

The Mycobacteria

Mycobacteria are members of the Actinomycete branch of the Gram - positive

bacteria. Other Actinomycete genera include the Nocardia, Corynebacterium and

Streptomyces(1). The name Mycobacterium stems from the mould-like appearance of

strains when grown as pellicles on nutrient broth. There are now at least 49 recognised

species of mycobacteria(1), however most of the energies associated with research into

mycobacteria are devoted to perhaps the best known members of the genus:

Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis (TB) in

humans; Mycobacterium bovis (M. bovis), the causative agent of tuberculosis in cattle

(and a range of mammalian species, including humans) and Mycobacterium leprae, the

agent responsible for the disease of leprosy. The mycobacteria have a genome of

approximately 4.5 million base pairs which contains a high G+C content (65.6%) and

around 4,000 genes, many of which encode enzymes involved in lipogenesis, lipolysis and

lipid transport(2). In fact, lipids may be responsible for one of the most characteristic

properties of the mycobacteria; that of acid - fastness(3). The ability to resist acid

mediated decolourisation after red fuchsin is not a property unique to mycobacteria but

1


was critical in the first identification of the bacilli(4, 5). Originally using Bismarck brown

and methylene blue, Robert Koch noted that “Under the microscope all the constituents

of animal tissue, that is, the cell nuclei and their products of disintegration appear brown,

while the tubercle bacilli, on the other hand, stain a beautiful blue”(4, 5). This technique

was modified by Ehrlich(6) who used red fuchsin and a mineral acid but was further

refined by the eponymous Ziehl (who optimised the acid decolourisation)(7) and Neelsen

(who combined Ehrlich’s red stain with Ziehl’s acid)(8).

The Mycobacterium tuberculosis Complex

The isolation of the tubercle bacillus in 1882 by Robert Koch(4, 5) was one of the

single most significant points in the history of microbiology, as it was also the first

isolation of any micro - organism in pure culture. However, it soon became apparent that

there were characteristic differences between the strains isolated from man, cattle and

birds. During the allocation of species in the 1890s, the mammalian tubercle bacillus was

named M. tuberculosis specifically to reflect its significance within the genus(9). Around

the same time it was noted that there were distinct morphological and cultural

differences between human and bovine isolates and that the bovine strains were more

virulent in rabbits(10).

In 1946, a further mammalian derivative of the tubercle bacillus was isolated from

voles(11) and became known as Mycobacterium microti(12). M. microti is primarily

pathogenic for rodents and demonstrates little or no pathogenicity in man or domestic

animals.

2


Further reclassification of the genus occurred in the 1960’s, when it was discovered that

strains initially thought to be M. tuberculosis isolated from patients in tropical Africa

resembled a heterogeneous group of isolates. These strains were phenotypically

different from M. tuberculosis and M. bovis and were named Mycobacterium

africanum(13).

Due to this close relationship, M. tuberculosis, M. africanum, M. bovis and M. microti are

known as the M. tuberculosis complex. A recent addition to the complex was a strain

isolated from human TB patients in Africa which proved to have a distinct phenotype,

different from the other members of the complex. This strain has been named

Mycobacterium canetti(14). Other members of the complex include M. caprae, M.

pinnipedii, M. suricattae, and M. mung.

Mycobacterium tuberculosis

Tuberculosis (TB) has been referred to as “one of a few severe communicable

diseases that may be described as true pestilences of mankind”(15). Often described as an

ancient disease, M. tuberculosis has been detected in the remains of Bison dating from

18,000 years ago(16) as well as in Neolithic human remains(17) and as Potts disease in the

spines of Egyptian mummies(18).

Today the tubercle bacillus continues to claim more lives than any other single infectious

agent with an estimated 8.7 million new cases of TB and 1.4 million deaths in 2011

alone(19). In recent years, the incidence of TB has risen drastically in both industrialised

3


and developing countries. Until 60 years ago, there were no anti - TB drugs, as was the

case for virtually all infectious diseases, yet in a recent survey by the World Health

Organisation (WHO), strains of M. tuberculosis resistant to at least a single anti -

tuberculous agent were documented in every country surveyed(20). This is compounded

by the emergence of strains resistant to both primary anti - TB drugs (isoniazid and

rifampicin), known as multi drug resistant TB (MDR - TB).

M. tuberculosis displays many characteristic features such as its slow growth, dormancy,

intracellular lifecycle and pathogenesis, and a complex cell envelope. With its generation

time of approximately 24 hours in synthetic medium or infected animals it is a difficult

organism to study and its long doubling time also contributes to the chronic nature of the

disease. This slow growth may also provide a selective advantage; where other

organisms outgrow their intracellular environment and kill their habitat, mycobacteria

slow their growth and convert into a so called “dormant” phase, thereby further limiting

their chances of destroying their hosts(21, 22).

M. tuberculosis is spread predominantly by the aerosol route, where droplet nuclei

containing 3 to 5 bacilli in a particle of <5 μm are inhaled and gain access to the alveoli(23-

25). Once inside the alveoli, the bacilli are engulfed by alveolar macrophages which

attempt to kill the organism via the respiratory burst and phagolysosome fusion(26, 27).

Virulent mycobacteria remain within phagosomes which fail to fuse with lysosomes, even

though mycobacterial phagosomes are almost identical to other early phagosomes(28),

and thereby avoid lysosomal degradation. This ability is dependent on viable bacilli, as

killed cells are rapidly destroyed(29, 30). By remaining in so - called “mycobacterial

4


phagosomes” the bacilli avoid destruction and also hinder the induction of adaptive

immune responses(31, 32).

Mycobacterium bovis

In his speech upon receiving the Nobel Prize in 1901, Emil von Behring stated “as

you know, tuberculosis in cattle is one of the most damaging infectious diseases to affect

agriculture”(33). Bovine tuberculosis (BTB) caused by M. bovis is a zoonotic disease that

has posed major animal health problems for the farming industry in England and Wales

since the 1930s(34) and affects cattle and other mammals worldwide.

M. bovis causes a disease in man which is clinically indistinguishable from M. tuberculosis

infection and it was thought previously that tuberculosis in domestic animals appeared

prior to its recognition in humans(35) and that M. tuberculosis was derived from M.

bovis(36). However, thanks to the completion of the genomes of M. tuberculosis(2) and M.

bovis(37), it has been shown that the pattern of deletions amongst members of the M.

tuberculosis complex suggests a derivation from a common ancestral organism(38, 39).

Many studies were performed comparing human and bovine strains, fuelled by the

incorrect statement “the human subject is immune against infection with bovine bacilli or

is so slightly susceptible that it is not necessary to take any steps to counteract risk of

infection” made by Koch to the British Congress on Tuberculosis in 1901(40). This

supposition was arrived at due to the relatively non - pathogenic nature of human

tubercle isolates in cattle and Koch assumed the strains were species - specific. The most

5


fervent voice of dissent came from the veterinary profession and the British Royal

Commission was appointed to study the issue. Research was funded by the Commission

between 1901 and 1911 and provided irrefutable proof that humans could be infected by

the bovine tubercle bacillus(41). The bovine bacilli became known as M. bovis; although

this name only became recognised in the literature considerably more recently(42).

The zoonotic importance of M. bovis cannot be underestimated. As far back as 1810, it

was reported that the incidence of tuberculous cervical lymphadenitis (also known as

scrofula) was higher in those children who were fed cow’s milk rather than breast milk(43).

The importance of M. bovis infection in humans was further highlighted in 1927 when a

study of 906 pulmonary and 202 extra - pulmonary cases of tuberculosis revealed that

67% of extra - pulmonary tuberculosis was caused by M. bovis(44). This zoonotic potential

is still of great concern today. Consumption of raw or unpasteurised animal products, or

direct contact with infected animals in slaughterhouses, plays a large role in zoonotic M.

bovis infection of humans in Africa and South America(45-47). Yet this is not just a problem

associated with less developed countries; recently Rodwell et al. have shown that as

much as 45% of all culture positive TB in children from Hispanic populations in San Diego

was caused by M. bovis(48).

6


Control of BTB In Great Britain

The control programme for BTB in Great Britain (GB) is a test and slaughter policy

which relies on the ability to identify M. bovis infected animals and their subsequent

removal and slaughter. This policy is in accordance with European Law which specifies

the requirements for testing and diagnosis of BTB(49). Previously, regular testing of cattle

occurred at intervals based on the prevalence of BTB in their geographical location

(parish testing intervals) which were determined each year based on the level of BTB in

the parish in the previous 6 years. However, on the 1st of January 2013, a new system

was introduced which replaced the use of parishes with county borders and split the

country into high and low risk areas based on disease prevalence. Animals within the

high risk area are tested annually whilst those in the low risk area are tested every four

years. Dividing the high and low risk areas is the ‘edge’ area, where BTB is not endemic

but animals are tested annually to enable any early detection of spread from the high risk

area. The high risk, low risk and edge areas are shown in figure 1:1.

7


Figure 1:1 - BTB testing areas shown for the year 2014 Available from http://ahvla.defra.gov.uk/documents/bovine-tb/pti-map.pdf.

8


Infection is identified using the single intradermal comparative tuberculin test (SICTT)

which enables the veterinarian to measure the delayed type hypersensitivity (DTH)

response to an intradermal injection of a crude preparation of mycobacterial antigens

known as purified protein derivative (PPD) or tuberculin.

In the early 1890’s, Bernhard Bang introduced the intradermal test as a diagnostic tool in

the control of BTB in Denmark. The ‘Bang method’ of testing consisted of repetitive six-

monthly use of the intradermal assay combined with physical separation of test - positive

and test - negative cows, and only culling cattle with “tuberculosis of the udder”(50). Bang

also introduced the pasteurisation of milk, buttermilk, and whey, to prevent transmission

of BTB via milk and milk products to calves long before pasteurisation of these products

for human consumption became standard(50). Following reports on the achievements of

the Bang method, it was accepted worldwide as the major tool in the control of BTB and

is still the basis of all control programs for BTB today.

It was Koch who developed what is now known as ‘old’ tuberculin, the reagent used as

the stimulating antigen in the early intradermal tests. While attempting to isolate the

“active principle of tuberculin”(51) for use as a treatment for human TB infection, Koch

subcutaneously injected heat killed samples of cultures into guinea pigs and noted the

presence of a characteristic skin reaction 24 - 48 hours later(52, 53). Despite not

recognising the importance of this reaction as a diagnostic tool, he introduced this

method to assess the potency of tuberculin preparations.

In the 1930’s a major improvement in the quality of the tuberculins, replacing Koch’s Old

Tuberculin, was achieved by the work of Florence Seibert. The use of a synthetic medium

9


and precipitation methods, facilitated a more reproducible and a large scale production

leading to the PPD reagents used today(54).

The advent of a compulsory skin test, after the voluntary Attested Herd Scheme of the

1950’s and 1960’s, coupled with compulsory slaughter of all test positive animals,

resulted in a significant drop in the levels of BTB in GB from an estimated 40% of all cattle

infected in 1934(34) to 0.41% in 1996(55). However, the original skin testing regimen was

not without its flaws.

The use of M. tuberculosis to produce tuberculin for use in cattle lead to a high

proportion of test - positive cattle which displayed no clinical evidence of disease when

examined post mortem. The introduction of the comparative test, using a tuberculin

derived from the avian mycobacterial species (M. avium), was based on pioneering

studies performed in the late 1930’s(56, 57). The use of avian tuberculin (PPD - A) adds a

significant degree of specificity to the skin test by allowing an assessment of any immune

priming caused by exposure to environmental mycobacteria. The skin test has remained

unchanged since with the exception of a switch from mammalian tuberculin, which was

produced using the M. tuberculosis strains DT, C and PN(58), to bovine tuberculin (PPD - B)

produced from an M. bovis strain isolated in England in 1948(59). It was found that the

tuberculin generated from the M. bovis AN5 strain was both more potent when used in

cattle and more specific(58).

The incidence of BTB in cattle in GB has shown a steady and continual increase since 1996

(figure 1:2), despite the unremitting implementation of control measures, possibly due to

10


the presence of a wildlife reservoir consisting primarily of the badger(55) (Meles meles).

Large scale trials have been performed to study the effect of culling badgers on the

incidence of BTB(55) which suggest that, while culling can reduce disease incidence in

cattle in the area of the cull, perturbation of the badger population leads to an increase in

disease occurrence in the surrounding areas(60). Within GB, BTB has spread drastically

since the Foot & Mouth disease outbreak in 2001 with the annual number of animals

slaughtered rising from a mean of 5,646 animals between 1996 and 2001 to an annual

mean of 29,504 between 2002 and 2012 inclusive(61).

Figure 1:2 - Incidence of BTB in GB since 1996 Bars represent number cattle subjected to SICTT; line shows number of cattle slaughtered. Available from:

http://www.defra.gov.uk/animal-diseases/a-z/bovine-tb/.

New Strategies for BTB Control

In 1996 Douglas Hogg MP, the then Minister of Agriculture, Fisheries and Food

commissioned an independent scientific review into Government policy on BTB. Chaired

by the Chief Executive of the Natural Environment Research Council, Professor John

11


Krebs, the review’s final report contained many recommendations, the first of which was

the development of a cattle vaccine; a strategy which was considered to be the best long

term option to control BTB(55).

The only licensed vaccine for use against TB (in any host species) is the attenuated M.

bovis strain bacille Calmette - Guérin (BCG). BCG is the most widely used vaccine in the

world today with more than 3 billion individuals immunised(62). The development of BCG

was the result of pathogenesis experiments at the Pasteur Institute in France where

Albert Calmette and Camille Guérin discovered that for successful infection of guinea pigs

the bovine tubercle bacilli needed to be emulsified(63).

While BCG is the only available vaccine for use against TB infection, reports of its

usefulness have varied. Overall, it is widely accepted that BCG vaccination significantly

reduces the risk of TB in humans by 50 %(64). However BCG daughter strains are not

identical(65, 66) and, across a variety of populations, the protective efficacy of BCG has

been reported anywhere between 0 % and 80 %, and usually in children and

adolescents(67). More recently it has been reported that BCG - induced protection may

last for up to 60 years in certain human populations(68) but other reports have shown that

2 doses of BCG are required to confer protection to children in Turkey(69) and a large

study performed in India involving patients covering a wide age range showed no

protective effect of 2 different BCG vaccines when compared with a placebo(70). The

reasons for this variable efficacy are not understood although many theories have been

proposed, such as interference of the immune response to BCG due to exposure to

environmental mycobacteria; differences between the BCG daughter strains used for

12


vaccination; the loss of protective antigens from BCG during passage and potential failure

in stimulating adequately balanced CD4+ and CD8+ T cell responses(64, 71). Other potential

confounders include variability in dose, route of administration, age of patients, genetic

variance between vaccinees and storage methods of the vaccine such as lyophilisation(64,

71). Clearly a better defined and tailored approach is needed to develop more efficacious

and promising vaccines.

A variety of strategies have been adopted in an effort to improve the efficacy of BCG

vaccination. One major route of investigation is the modification of BCG at the genomic

level such that it overexpresses known tuberculoid antigens such as 85B (Ag85B). For

example, the vaccine candidate rBCG30, which overexpresses Ag85B, has been shown to

be more protective than it’s parental strain in animal challenge studies(72, 73).

A second improvement strategy has been to enhance priming of CD8+ T cells, rather than

primarily targeting CD4+ T cells. An elegant strategy developed by Kaufmann has been to

enhance the ability of BCG to escape from phagocytic endosomes, thus allowing

endogenous antigen processing and presentation in the context of the major

histocompatibility complex (MHC) I. Inclusion of a cytolysin gene (hly) from Listeria

monocytogenes into the BCG genome, along with deletion of the BCG urease gene ureC

enables the vaccine to puncture the membrane of the early endosome and escape into

the cytoplasm(74). Not only does this enable greater endogenous antigen processing and

MHCI mediated presentation, but bacterial escape from the endosome can lead to

apoptosis in the infected cell and hence allow cross presentation of exogenous antigen to

13


naïve CD8+ T cells(75). This approach has led to enhanced protection over the parental

strain and was also safer in immunocompromised SCID mice(76).

Other potential improvements to BCG include the addition of superoxide dismutase to

the genome to enhance BCG survival and granuloma formation(77) and the reintroduction

of genes that were lost from BCG during its attenuation such as the RD1 locus(78). This

latter approach is not without criticism, for fears that it may increase the virulence of

BCG(79). Alternatives to modification or enhancement of BCG itself are also being

assessed. Generation of a live attenuated M. tuberculosis vaccine is a possibility,

although there is concern over the potential for these strains to revert to a virulent

phenotype(80).

The use of antigens which activate T cells in previously infected subjects may also provide

novel vaccines. The search for antigens has identified a raft of potential vaccine

candidates, some of which have been formulated into recombinant fusion proteins. The

vaccine candidate Hybrid-I, consisting of Ag85B and the immunodominant Early Secreted

Antigenic Target 6 kDa (ESAT - 6), has been shown to be as protective as BCG when

formulated with an adjuvant(81, 82). The use of ESAT - 6 as an immunising antigen is

controversial as it is also the primary basis of the new generation of diagnostic tests,

which may be compromised by its use(83).

Antigenic proteins such as Hybrid-I are promising vaccine candidates but there is little

improvement in protection over traditional BCG vaccination. However, these candidates

may be of significant use in a highly productive area of research - the heterologous prime

14


- boost strategy. Using an initial priming vaccine and boosting with a more defined

subunit has the advantage that the boost preferentially expands TB - specific memory T

cells generated by the initial priming immunisation. Using BCG as a primary vaccine

allows the potential to boost with any of the subunit candidates currently under research,

such as Hybrid-I, however these boosting antigens may not stimulate sufficient CD8+ T

cell responses.

A popular trial strategy used a recombinant, non - replicating virus presenting antigenic

subunits on its surface as the boost to the BCG prime. The use of the poxvirus Modified

Vaccinia Ankara (MVA) which expresses Ag85A, known as MVA85A, as a boosting agent,

after priming with BCG, has been shown to induce greater levels of antigen specific CD4+

and CD8+ T cells as well as greater protection against challenge when compared to either

BCG or the MVA85A alone(84). Similar responses have been seen in human trials with this

regimen where immunisation with one of the vaccines generated only short - lived

antigen specific immune responses but the prime - boost strategy generated longer lived,

stronger responses(85). However in a phase 2B clinical trial where 2,797 BCG vaccinated

South African infants were boosted with MVA85A, only 2 % of participants were

protected against M. tuberculosis infection(86). The authors note that the levels of antigen

specific CD4+ T cells in the study participants was as much as 90 % lower than in previous

studies performed with adults(87, 88) and suggest that understanding the mechanisms

behind expanding the appropriate T cell populations may be critical to the development

of an efficacious human vaccine.

15


As reported in humans(67), estimates of protective efficacy of BCG in cattle have ranged

between 0 % and 70 %(89-94) and heterologous prime - boost strategies also show promise

in cattle(95, 96). Similar to work in humans, a variety of alternative strategies have been

tried in cattle. DNA vaccines expressing mycobacterial antigens (including MPB70,

MPB83, Hsp60 and Hsp70) have been developed and trialled with generally inadequate

results(97, 98). However, one study reported significant protection using a DNA vaccine

which encoded ESAT - 6 as well as the co - stimulatory molecules CD80 and CD86, usually

found on dendritic cells (DC)(99). DNA vaccines have proven more successful when

applied in a heterologous prime - boost strategy based upon a BCG prime(97).

Interestingly, a subsequent follow up study from the same authors demonstrated no

difference in protection if the DNA vaccine was used to prime and BCG used as the

boosting antigen(100). Vital work performed by Vordermeier et al. compared several

vaccination strategies in cattle using BCG and boosting with either MVA85A or a

recombinant adenovirus also expressing Ag85A (Ad85A) and demonstrated greater

protective efficacy with the heterologous prime - boost strategies(96). Critically, this was

the first time any enhanced protective effect was shown in a natural host of

mycobacteria.

Another potential method for successful vaccination of cattle is administration of BCG via

the oral route. Studies performed in New Zealand demonstrated that this method

conveyed similar protection to experimental challenge as subcutaneous immunisation,

however the dose had to be increased and the vaccine had to be administered in a lipid

formulation(101). Unfortunately, reducing the dose of BCG used in these experiments

renders this system unprotective(102) and the protective efficacy cannot be increased by

16


co - administration of mycobacterial proteins as heterologous prime - boost antigens(103).

Interestingly, all oral vaccines trialled have required formulation in a lipid matrix to confer

protection(104-107). Not only do these lipid formulations enhance BCG survival in the gut,

but they can play a role in enhancing the host immune response(108). Several commercial

lipid adjuvants exist which have been shown to enhance Th1 responses or vaccine

efficacy(109-111) and the addition of mycobacterial lipids into these adjuvant formulations

has been further shown to enhance the protective activity of vaccines. The most studied

mycobacterial adjuvants have been the cord factors. Trehalose dimycolate (TDM) has

been shown to enhance long term protection of mice when administered as an adjuvant

for the boost in a heterologous prime - boost strategy(112) and use of the synthetic

analogue trehalose dibehenate (TDB) as an adjuvant has been shown to activate Th1 and

Th17 responses via the macrophage inducible Ca2+ - dependant C - type lectin

(Mincle)(113). The polar lipid mono - mycolyl glycerol (MMG) has also been formulated

into an adjuvant which has been shown to be capable of activating DC and driving Th1

responses(114, 115). These advances clearly highlight the role of lipids in modulating

immune responses and their potential applications as adjuvants or vaccine candidates.

Mycobacterial Lipids

Biologically, the mycobacteria are considered to be Gram positive as they possess

a single peptidoglycan cell wall. What is unusual about the mycobacterial cell wall is that,

rather than proteins and carbohydrates found in many other bacteria, most of the

molecules attached to it are lipids(116). In fact, it has been estimated that as much as 60%

of the dry weight of mycobacteria is cell wall associated lipid(117) and these lipids are

17


widely believed to be important in directing the interactions between the pathogen and

its host(118, 119). Fractionation of the mycobacterial cell envelope is difficult as the

envelope is physically strong and fractions tend to adhere to one another(120), however

pioneering work by Brodie et al., using rapid growing mycobacterial species, lead to the

ability to isolate the plasma membrane and fractionate the cell wall(121).

While the plasma membrane appears essentially the same as found in other Gram

positive bacteria, there are some distinct membrane bound components not found in

other bacterial genera, primarily lipomannan, lipoarabinomannan (LAM) and

phosphatidylinositol mannosides (PIMs)(120). Freeze fracture studies of the mycobacterial

plasma membrane show a typical fracture plane between the membrane surfaces, with

integral membrane proteins embedded in these layers(122), and no evidence of a second

outer membrane, such as those found in Gram negative bacteria. However a second

plane of fracture has been identified which is associated with carbohydrate molecules(123)

that may include LAM and PIMs(120).

Much time and effort has been invested in developing a comprehensive model of the

mycobacterial cell envelope. Thin - section transmission electron microscopy of

mycobacteria reveals a plasma membrane surrounded by a thick electron - transparent

periplasmic space. External to this is an electron - dense layer which, unlike the

peptidoglycan found in most Gram positive bacteria, is believed to be composed of a

peptidoglycan and arabinogalactan complex. Attached to this is another electron -

transparent layer of mycolic acids(124). A final electron - dense outer layer composed of

carbohydrates and protein surrounds the bacterium and may also contain lipids(125-127).

18


This lipid - rich envelope is unusual in that it forms a permeability barrier and may be a

bilayer in composition, similar to the outer membrane seen in Gram negative

bacteria(128).

The outer membrane, and the arrangement of the lipids therein, has been the study of

enormous research and many models have been proposed(128-132). It is believed that

mycolic acids are covalently bound to the external arabinogalactan portion of the cell wall

and an outer membrane of various lipids surrounds this. Lipids found in this outer

membrane may be glycolipids, such as trehalose monomycolate (TMM) and TDM,

phospholipids, phthiocerol dimycocerosates and other species - specific lipids(128-132). The

model is illustrated in figure 1:3 and the structures of some of the outer lipids are shown

in figure 1:4.

19


Figure 1:3 - Model of the mycobacterial cell wall. Symbols do not denote specific molecules. PI: phosphatidylinositol.

20


Figure 1:4 - Structures of some common outer envelope lipids found in virulent mycobacteria (A) Phosphatidylinositol dimannoside (PIM2); (B) Diacylated phosphatidylinositol hexamannoside (Ac2PIM6); (C)

Phthiocerol dimycocerosate (PDIM); (D) Trehalose-6-6’-dimycolate (TDM); (E) Phosphatidylinositol (PI); (F) Phosphatidylethanolamine (PE).

The structure of the cell wall and its outer envelope are critical to the stability of the cell.

It is well documented that mutations affecting either the length of mycolic acids(133) or

their modification(134-136) can alter the phenotype of the bacterial cell leading to altered

colony morphology and persistence in host cells(137) or alter the permeability and lability

of the outer membrane(138). In fact, genetic removal of mycolic acids from

Corynebacterium glutamicum caused complete removal of the outer membrane(127, 139)

and even renders the mutant bacteria more permeable to drug entry(140).

21


Immune Recognition of Lipids

Dendritic cells (DC) are a group of bone marrow derived leucocytes, found in

many tissues which serve several functions but, perhaps most importantly, initiate

adaptive immune responses(141). Originally identified in 1973 by Steinman & Cohn(142), DC

are highly specialised for antigen uptake, processing and presentation to T cells and are

considered to be the only antigen presenting cells (APC) capable of presenting antigen to,

and activating, naïve T cells(143). These cells are considered to epitomise the so called

“professional” APCs(142). Their dendritic membrane extensions maximise the surface area

of the cell and the membrane is replete with MHC, adhesion and costimulatory

molecules(141, 142). Tissue localisation and different routes of derivation from the bone

marrow mean that there are several types of highly specialised DC(144).

So - called immature DCs, primarily resident in peripheral tissues, are highly efficient

phagocytes capable of engulfment and presentation of a huge variety of antigens(145).

Activation of these “sentinel” cells is driven via a signal, which may consist of physical

tissue insult (and the consequent release of TNFα and IL - 1), antigen uptake or contact

with molecules containing pathogen - associated molecular patterns (PAMPs)(142, 146, 147).

Activated DCs exit tissue via the draining lymphatic system and migrate to the draining

lymph nodes. Migration is mediated by chemotactic gradients and this migratory stage is

often characterised by increased expression of the chemokine receptor CCR7(148-150). The

chemokines CCL19 (MIP - 3β) and CCL21 (6Ckine) are ligands for CCR7 and their

production by lymph node resident stromal cells is known to be important in directing

22


naïve T cells and migratory DCs to the lymph node(151). However, whether expression of

both of these chemokines is essential for DC migration remains unclear(152-154).

Upon arrival at the lymph node, DCs are capable of stimulating and modulating the T cell

response, a process believed to be driven by the innate interaction between DCs and

pathogen and the subsequent production of IFNα and IL - 12(155-157). These DCs are

primarily characterised by their increase in expression of MHCII molecules on the cell

surface(158), as well as other co - stimulatory molecules such as CD40, CD80 and CD86(141,

159).

Lipid Modulation of Innate Immune Responses

The role of macrophages in protection against tuberculous infection was

discovered over 60 years ago(160-162). Much work was performed in the 1980s which lead

to a greater understanding of the interaction between the macrophage and other host

immune components including a method of macrophage mediated bacterial killing using

hydrogen peroxide(163) and the critical role of IFNγ in the activation and enhancement of

macrophages and their killing(164, 165). Since then the interaction between mycobacterial

species, usually M. tuberculosis or BCG, and APCs has been heavily studied. Both DCs and

macrophages can interact with mycobacteria using an array of receptors such as

complement receptors (CR) 1, 3 and 4; and pattern recognition receptors (PRRs) including

the scavenger receptor, the Toll - like receptors (TLRs), nucleotide binding oligomerisation

domain containing (NOD) - like receptors and surface bound lectins such as the mannose

receptor (MR) and the DC-specific intercellular adhesion molecule - 3 grabbing

23


nonintegrin (DC - SIGN)(166). Ligation of different receptors drives different effector

functions, some specifically promoting phagocytosis (e.g. scavenger receptors) and others

triggering non - phagocytic maturation or activation (such as the TLRs). It is the selective

and multiple ligation of these receptors which dictates the effector function of the

APC(167, 168).

Of course, a successful pathogen is one which is capable of, in part at least, avoiding or

subverting the host immune response. It was originally hypothesised in the 1970s that

M. tuberculosis had the ability to avoid killing by the macrophage(29, 169). In fact it has

been suggested that the typical immunopathology associated with pathogenic

mycobacterial infection (the granuloma) may be beneficial to the bacilli(170).

Nevertheless, the interface between the bacilli and their host is pivotal in defining the

progress of infection, and those cells which interact with the pathogen both initiate and

shape the response. Immune recognition of M. bovis (as well as M. tuberculosis) is

mediated by receptor : ligand interaction and lipids, being so abundant in the

mycobacterial cell envelope, are likely to be central in this contact.

Interaction between mycobacteria and a variety of mammalian PRRs has been well

documented(171-174). Both macrophages and DC have been shown to recognise a variety

of lipid antigens via TLR2 including AraLAM(175), lipomannan (from both M. tuberculosis

and BCG)(176), PIM2(175) and PIM6

(177). Other PRRs, especially C - type lectins such as the

mannose receptor (CD207), DC - SIGN (CD209) and Dectin - 1, have also been

documented as playing important roles in sensing tuberculous infection.

24


Macrophage phagocytosis of M. tuberculosis is primarily mediated through the mannose

receptor and usually leads to an anti - inflammatory response(178, 179). M. tuberculosis

derived mannosylated LAM (ManLAM) has been shown to bind to the mannose receptor

and is capable of inhibiting IL - 12 production(180) and this interaction is also a key step in

blocking phagosome - lysosome fusion(181).

DC - SIGN is usually found on the surface of DC and is known to bind a range of

mycobacterial lipids including ManLAM and lipomannans(182, 183). This has been shown in

vivo and it has been suggested that interaction with DC - SIGN may allow mycobacterial

entry into a DC, where it remains during DC migration to the lymph nodes(182). More

recently, Doz et al.(171) have shown that diacylated lipomannans are capable of inhibiting

both cytokine and nitric oxide production in LPS - activated macrophages and this

property was assigned to interaction with either DC - SIGN or the mannose receptor(171).

Although Dectin - 1 is more commonly associated with pattern recognition during fungal

infection, recent studies have suggested a role in TNFα production in M. avium - infected

macrophages(184). In another study, Dectin - 1 dependant signalling was been shown to

be involved in the production of IL - 12p40 and IL - 12p70 by splenic DC in response to M.

tuberculosis(185). Despite these studies no specific mycobacterial lipid ligands for Dectin -

1 have been found.

Innate recognition of mycobacterial antigens may not be limited to the cell surface. NOD

like receptor (NLR) 2 has been shown to synergise with TLR2 to increase nitric oxide and

TNFα production(186) whilst removal of NOD2 from murine macrophages and DC impairs

cytokine and nitric oxide production in response to live M. tuberculosis(187). This study

25


also showed that the arabinogalactan - peptidoglycan cell wall core of M. tuberculosis

induced TNFα and IL - 12p40 production via NOD2 in murine macrophages (187).

Lipid Recognition by Adaptive Immunity

Lipids have been shown to mediate a range of effects on lymphocytes. Molecules

such as LAM are known to be chemotactic for T cells when purified from either virulent or

attenuated M. tuberculosis(188). Interestingly, supernatants from macrophage cultures

only retain this chemotactic activity when infected with virulent M. tuberculosis

suggesting differences between the strains in their ability to export LAM(188). ManLAM

has also been shown to supress T cell proliferation when added to T cell clones specific

for other antigens(189) and also to supress T cell activation as measured by reduced mRNA

expression of IL - 2, IL - 3, GM - CSF and the IL - 2 receptor α chain(190). The discovery that

T cells could recognise lipids if the antigen is presented in the context of CD1 was a

significant breakthrough(191). It is now known that, within the human genome at least,

there are 5 CD1 molecules: CD1a - e. These are split into 3 groups based on their

sequence homology; group 1 consists of CD1a, b and c, group 2 just CD1d and group 3

contains CD1e.

CD1a is found on DC and differs from other group 1 CD1 molecules in that it is

constitutively expressed at high levels on human Langerhans cells(192) and has been

shown to be able to present some mycobacterial lipids to CD8+ αβ T cells(193). CD1a has

also been found to present the mycobacterial lipopeptide didehydroxymycobactin(194)

and is capable of activating CD8+ αβ T cells(195). As the precursor to mycobactin, an iron

26


chelating agent critical for survival within the macrophage, levels of

didehydroxymycobactin increase during intracellular growth(196) and this may be an

important mechanism for recognition of infected cells.

The second group 1 molecule CD1b is expressed by activated monocytes and DC and

presents most of the identified mycobacterial lipid antigens(197) and is unique amongst

CD1 molecules in that the structure of the binding cleft allows for binding of lipids of a

large range of sizes(198-200). The first CD1b restricted mycobacterial antigen identified was

mycolic acid, which was shown to stimulate T cells(191), and all subsequent mycolic acid

based lipid antigens are presented through CD1b thanks to its ability to accept long acyl

chains into the binding pockets.

In fact, the mycolic acid structure may be responsible for the immunstimulatory activity

of other lipids as many are based on the same backbone. For example, GMM consists of

a glucose residue attached to a mycolic acid and is capable of stimulating specific T

cells(201). GMM loaded CD1b tetramers have been shown to label T cells from M.

tuberculosis infected patients(202) suggesting an expansion of lipid responsive T cells in

response to infection. These tetramer labelled cells were found to be CD4+ and express

limited diversity in their TCR repertoire and further analysis demonstrated expansion of

these cells in patients latently infected with M. tuberculosis(203). Interestingly, the authors

also identified that the restricted TCR repertoire was associated with high levels of

tetramer labelling and found that tetramer - intermediate labelled cells expressed a more

diverse TCR repertoire suggesting the presence of other CD1b restricted mycobacterial

lipids(203).

27


GroMM is another example and consists of a glycerol moiety attached to a mycolic acid

structure and has been shown to drive T cell proliferation and IL - 2 production(204).

Another family of CD1b presented lipids are those containing PI such as PIM and LAM(205).

Both LAM and LM, which lacks the arabinose molecules, are capable of stimulating

specific T cells through CD1b although some discrimination is made between molecules

from different bacteria(205). Despite the large binding pockets of CD1b, lipoglycans

require internalising and processing before they are bound(206). For example, the

hexamannsoylated PIM6 is known to stimulate T cells in the context of CD1b but only

after partial digestion involving CD1e(207). It has since been shown that CD1e selectively

assists an α - mannosidase based on the degree of acylation of the PIM molecule(208).

Sulphoglycolipids are also presented by CD1b(209) but these molecules are not present in

M. bovis (210, 211).

The final group 1 CD1 molecule is CD1c which is unique in that the binding cleft allows the

hydrophilic component of a bound antigen to protrude and it is thought that this may

interact with the T cell receptor(212). While CD1c cycles through deep endosomal

compartments where lipid binding proteins are present in a similar fashion to CD1b and

CD1d, it also cycles through early endosomal compartments where the pH is higher and

no lipid chaperone proteins are present(197) and it is thought that the partially open

structure of CD1c may allow for antigen binding to happen in this environment(197).

CD1c has been shown to present a variety of lipids of a range of structures(213-215) and the

role of CD1c in mycobacterial infection has been well characterised. Experiments with T

cell lines meant that it was initially thought that CD1c presented branched chain

phospholipids such as mannosylated phosphomycoketide(213) however use of the DN6 T

28


cell line showed responses to unglycosylated molecules revealing the specificity of the

cell line to be naked phosphmycoketide(216). Further, CD1c tetramer analysis showed only

unglycosylated molecules were recognised despite the ability of cells to respond to

glycosylated molecules when presented by APC suggesting that antigen processing must

be removing the glycosyl residues(216).

Unusually, CD1 molecules are also known to be ligands for both αβ and γδ T cells. Initially

γδ T cells were found to be CD1c responsive(217) but studies with duodenal cells have

since shown this population to be responsive to all group 1 and group 2 CD1

molecules(218) despite a limited TCR repertoire. The use of CD1d tetramers has identified

a sulphatide responsive γδ population in peripheral blood which express the same limited

TCR repertoire(219). While little is known about γδ T cell ligands, CD1d has been shown to

be expressed in locations where this restricted γδ T cell population exists(220-222) and

activated cells have a Th1 effector phenotype and produce granulysin(217) hence CD1d

may constitute a legitimate γδ T cell ligand.

Found on the surface of most haematopoietic cells, CD1d is the only group 2 molecule

and one of the best studied of the CD1 family. Crystal structure analysis performed using

antigen - loaded CD1d bound to a TCR has shown that the molecule binds lipids with acyl

chains of specific lengths(223). Levels of CD1d are highly variable and alter in response to a

range of microorganisms, the receptors ligated and the presence of different soluble

factors. M. tuberculosis infection or exposure to mycobacterial lipids has been shown to

upregulate CD1d expression on bone marrow - derived macrophages(224) whilst infection

of monocytes prevents the upregulation of CD1d in cells that differentiate into DC(225).

29


CD1d restricted T cells are classified into 2 groups; the invariant NKT cells and the non -

invariant CD1d restricted T cells. Invariant NKT cells in mice and humans express only a

single TCR α chain and only a small selection of β chains and are characterised by their

high affinity recognition of α galactosylceramide (αGalCer)(226) but these cells are highly

conserved in other species(197). CD1d restricted invariant NKT cells are known to

recognise certain non - microbial lipids including PI and PE, albeit with low affinity in

comparison to αGalCer(227), and one study has suggested that these cells are also capable

of recognising mycobacterial PIM4 extracted from BCG(228).

The non - invariant CD1d restricted T cells resemble other, more traditional T cells and

are involved in classical adaptive responses(197). These cells have been shown to

recognise DPG and PI from M. tuberculosis(229).

Group 3 CD1 molecules consist solely of CD1e which remains within the cytosol of DC and

is not externalised(230). CD1e acts as a lipid binding protein and mediates the transfer of

lipids to CD1b(231) and also in the degradation of some large lipid molecules, such as PIM6,

as discussed above(207, 208).

The Aims of this Study

Acquired cellular immune responses are utterly dependent on the initial

interaction between the pathogen and the host’s innate immune system and this first

point of contact for M. bovis is likely to be an interaction with either alveolar

macrophages or dendritic cells in the host lung. It is widely accepted that the interaction

between the phagocyte and the external molecules of the bacilli play a significant role in

30


the establishment of infection and, hence, the outcome of disease(118, 143, 159, 166, 232-238).

Cell - mediated immunity is essential in developing both protective and pathological

immunity to tuberculosis, as pulmonary macrophages alone are insufficient to control

infection. Rather, they act as a refuge for the bacilli which are capable of inhibiting

phagosome - lysosome fusion and thereby avoiding destruction(239, 240). In humans,

granuloma formation usually contains the infection(241) leading to a state of so - called

dormancy, or latency, although infection is rarely eliminated. However, in cattle there is

considerable debate about the presence of latent infection(242).

As previously discussed, the production of IL - 12 by DCs and the subsequent Th1

polarisation of the cell - mediated response is essential for IFNγ production and

macrophage activation(243) and the bacilli : antigen presenting cell interaction is perhaps

the most important event that occurs during the infection process(170). Given the high

levels of lipid found on the mycobacterial cell wall, it is highly likely that these lipids will

play an important role in immune recognition of the pathogen. In fact, many lipids have

already been shown to stimulate potent immune responses(115), some have even been

associated with immune suppression and hypervirulence(244) and immunostimulatory

lipids have even been used to develop Th1 polarising adjuvants(114).

Yet there is a distinct lack of published information regarding the interaction of the

virulent pathogen and it’s hosts APCs. A search of PubMed using the relevant terms

returns very few results and none of them discuss pathogen derived lipid in host - derived

APCs. For example, Andersen et al.(115) demonstrated upregulation of CD40, CD86 and

HLA - DR and the production of IL - 6 and TNFα by MDDC derived from human

31


macrophages but either extracted lipids from M. bovis BCG rather than the virulent

human pathogen M. tuberculosis or used chemically synthesised MMG. Bovine specific

work performed by Hope et al. made use of bovine monocytes in the generation of

MDDC but these cells were stimulated with a synthetic lipopeptide(233) rather than a

pathogen - derived lipid antigen. In the study performed by Reed et al.(244), the authors

showed that blockage of synthesis of the phenolic glycolipid (PGL) correlated with

increased secretion of TNFα, IL - 6 and IL - 12 by the host, and removed the “hyperlethal”

phenotype displayed by the bacilli, however this was shown only in the murine model.

Immunologically active bacterial lipids play a major role in the outcome of tuberculous

infection and have a range of potential practical uses. The use of specific lipids from BCG

and M. tuberculosis to enhance protective efficacy or duration of immunity of vaccines

has been demonstrated in a variety of model systems(112-115).

Further, given the ability of lipids to traffic more freely than proteins between

macrophage intracellular compartments(245) and the ability of lipids to enter directly into

the membranes of APC(246), it is possible that lipid molecules themselves could confer

protective immunity. A potential advantage of this approach to vaccine development is

that CD1 molecules are highly non - polymorphic and CD1 - presented lipids are not

subject to large structural changes(247) as well as the fact that CD1 - restricted T cells are

known to exhibit anti - microbial activity(213, 248). These data suggest that specific lipid

molecules could be used as adjuvants or as vaccine candidates for cattle however more

needs to be known about how lipids interact with bovine immune cells.

Lipids such as mycolic acids and LAM have also been investigated as highly specific

diagnostic reagents in humans(249-251) and similar methods could be applied to cattle.

32


Therefore, this study addresses the hypothesis that lipids from M. bovis constitute a

source of molecules that could be developed into tools which could contribute to the

development of control measures for BTB. These may include subunit vaccine candidates

acting as antigens, or biological adjuvants, immunostimulators and specific diagnostic

reagents. As a first step towards these goals, this thesis aims to identify and

immunologically characterise M. bovis - derived lipid preparations and compounds by

pursuing the following objectives:

• Develop methods to isolate and characterise lipid moieties from M. bovis,

• Assess the effect of M bovis lipids on bovine APC to identify potential

immunomodulatory activity which could be exploited in the development of novel

adjuvants,

• Identify the lipid targets of the bovine adaptive immune response which could be

used as innovative vaccine candidates.

33

Chapter Two Materials & Methods

Chapter Two

Materials & Methods

Preparation of Bacterial Isolates for Lipid Extraction

Mycobacterum bovis strain AF 2122/97 was grown in Middlebrooks 7H9 medium,

prepared using 4.7 g 7H9, 2 ml glycerol and 0.5 g Tween 80 in 800 ml of reverse osmosis

purified water (roH20). This mixture was stirred until homogeneous and autoclaved at

121 oC for 30 minutes. After cooling, 100 ml of a sterile 10x sodium pyruvate stock was

added along with 100 ml of sterile Middlebrooks albumin / dextrose / catalyse (ADC)

enrichment broth.

Initial starting cultures were performed in 10ml volumes in static flasks at 37 oC within an

ACDP Containment Level 3 (CL3) facility. Large quantities of bacterial cells were grown in

100 ml rolling culture flasks inoculated with 1ml of starting culture and incubated until

mid - log phase, defined as an optical density (at 600 nm wavelength) of 0.6 - 0.8.

To harvest cells, mid - log phase cultures were decanted into sterile 50 ml Falcon tubes

and spun at 2,500 g for 5 minutes. Supernatants were decanted and cell pellets were

34


washed twice in sterile milliQ purified water. Pellets were suspended in 5ml sterile milliQ

water and stored at -80 oC until being killed and removed from the CL3 facility. Cell

pellets were thawed before killing and heat killed in a water bath at 80 oC to 90 oC for

between 1 and 2 hours. Finally, heat killed cell pellets were freeze dried prior to

extraction of lipids.

M. bovis AN5 was sourced from the Tuberculin Production Unit at AHVLA Weybridge.

Bacterial cells were grown in an ACDP CL3 laboratory as a surface pellicle on glycerol rich

Bureau of Animal Industry (BAI) medium containing 14 g of L - asparagine, 1.5 g of

dipotassium hydrogen phosphate, 0.74 g of sodium citrate, 1.5 g of magnesium sulphate,

0.3 g of ferric sulphate, 0.08 g of zinc sulphate, 0.008 g of manganese chloride, 0.00138 g

of cobaltous chloride, 10 g of glucose and 100 g of glycerol litre-1.

The pellicle was autoclaved at 134 oC for 1 hour before being freeze dried prior to

extraction of lipids.

Extraction of Crude Free Mycobacterial Lipid

The extraction method used in this thesis has been previously described(252).

Freeze dried bacterial cells were added to a mixture of 400 ml CH3OH and 40 ml 0.3 %

aqueous NaCl. A further 440 ml of petroleum ether was added, the top of the vessel

covered and mixed for 12 to 16 hours. The mixture was decanted into centrifuge bottles

and spun at 4000 g for 10 minutes to pellet the bacterial cells. The upper, non - aqueous

phase was removed by pipetting and stored. To the lower, aqueous phase a further 440

ml of petroleum ether was added and the mixture stirred for 2 hours before being spun

35


again, the non - aqueous layer removed and pooled with the first. The petroleum ether

was removed from these extracts using a rotary evaporator with cold finger condenser

and the lipid transferred to a pre - weighed glass tube in 4 : 1 CHCl3 : CH3OH. The CHCl3 :

CH3OH mixture was evaporated using a heating block and a stream of N2 gas leaving dried

lipid in the pre - weighed tube which was subsequently weighed again to determine the

mass of apolar lipids extracted.

Extraction of the polar lipids was performed by adding 212 ml CHCl3, 236 ml CH3OH and

72 ml 0.3 % aqueous NaCl (520 ml of 9 : 10 : 3 mixture of CHCl3 : CH3OH : NaCl). This

mixture was stirred for 12 to 16 hours before being passed through double Whatman 91

240 mm filters papers to collect the cells. Once dried on the filter, the cells were re -

extracted twice using 170 ml of a 5 : 10 : 4 mixture of CHCl3 : CH3OH : NaCl. After a final

filtration to remove cells and cell debris from the polar phase, 290 ml of both CHCl3 and

NaCl were added to polar phase which was mixed for one hour and allowed to split in a

separating funnel. The lower, aqueous, phase was removed and dried in a rotary

evaporator. Final polar lipid mass was ascertained as described for the apolar petroleum

ether extracted lipid fraction. The process is illustrated in figure 2:1.

36


Figure 2:1 - Schematic representation of the extraction of free mycobacterial lipids.

Analysis of Lipid Fractions by 2D Thin Layer Chromatography

Aluminium backed silica gel 60 F254 TLC plates were cut into approximately 6 cm

squares and 100 μg of lipid extract was spotted onto the plates using glass micro -

capillary pipettes. Plates were dried at 80 oC in an oven for 10 minutes before running.

Appropriate solvent mixtures were prepared in 100 ml volumes as previously

described(252) and TLC tanks were equilibrated using blotting paper before plates were

37


run. The solvent systems used in all 5 TLC systems are shown in table 2:1 and plates were

run as indicated.

TLC plates were dried for 10 minutes at 80 oC between each run and before staining to

ensure that all residual solvent was removed.

Staining was performed using either a 5 % solution of molybdophosphoric acid (MPA) in

100 ml of 95 % v/v ethanol in water (MPA) or a 1.5 % w/v solution of ninhydrin dissolved

in 100 ml n - butanol to which 3 ml glacial acetic acid was added. Stains were sprayed

onto TLC plates which were subsequently charred using a hot air gun before being

photographed or scanned.

Table 2:1 - Solvent systems for TLC analysis of mycobacterial lipids (adapted from Dobson et al.(252))

Solvent System

Run Direction Components Runs

Fractions Analysed

Lipids Resolved

A 1 petroleum ether : ethyl acetate (98 : 2) 3 Apolar PDIM,

TAG, MQ 2 petroleum ether : acetone (98 : 2) 1

B 1 petroleum ether : acetone (92 : 8) 3 Apolar ATs, PGL,

MMG 2 toluene : acetone (95 : 5) 1

C 1 chloroform : methanol (96 : 4) 1 Apolar PGL,

MMG 2 toluene : acetone (80 : 20) 1

D 1 chloroform : methanol : water (100 : 14 : 0.8) 1 Apolar & Polar

CF, SL, GMM 2 chloroform : acetone : methanol : water (50 : 60: 2.5 : 3) 1

E 1 chloroform : methanol : water (60 : 30 : 6) 1 Polar DPG, PE

PI, PIM 2 chloroform : acetic acid : methanol : water (40 : 25 : 3 : 6) 1

38


Thin Layer Chromatography Densitometrical Analysis

High resolution uncompressed TIFF images were loaded into Quantity One

analysis software and the gradient tool used to select individual lipid spots. Automatic

and manual separation of the spots from the background colour was found to be optimal

when the TLC images were false coloured using the “spectrum” option. A block selection

of the entire plate was also made to include all lipids, including those present either at

the origin or solvent front (figure 3:5). Calculation of the volume of each spot was

performed by multiplying the intensity of the spot by its area in mm2. The intensity of

each spot was calculated as a percentage of the total plate.

Preparation of Lipid Antigen Suspensions

Suspensions of all lipid antigens were prepared in an aqueous phase for use in cell

culture experiments after first removing any CHCl3 : CH3OH by evaporation using an N2

gas stream. Complete RPMI was added to the dried lipid and the mixture was then

heated to 80 oC for 5 minutes followed by sonication for 5 minutes. This heating and

sonicating cycle was repeated twice. A visual check was made of lipid preparations; were

any lipid visible to the naked eye either on the glass or in solution, another round of heat

and sonication was performed. Apolar and polar lipids were used at 20 µg ml -1 for 12 -

16 hours.

39


Lipid Purification by 1D Thin Layer Chromatography

Thirty milligrams of crude free mycobacterial lipid was streaked onto the long side

of 10 cm x 20 cm plastic backed silica gel 60 F254 TLC plate. Plates were run in an

equilibrated 14 : 6 : 0.8 mixture of CHCl3 : CH3OH : H2O and dried with warm air before

being sprayed with a solution of 0.01 % 1, 6 - diphenyl - 1, 3, 5 - hexatriene (DPH) in a 9 :

1 mixture of petroleum ether : acetone. Staining was visualised under long wavelength

ultra - violet light (366 nm; UV - A) and visible bands were marked with pencil. A

representative plate is shown in figure 2:2.

Once bands had been marked, plates were washed in toluene to remove DPH and

confirmation of removal was made by visualising the plate under 366 nm UV light. Bands

were then scraped from the plate and collected into individual glass tubes containing 5 ml

of a 2 : 1 mixture of CHCl3 : CH3OH and mixed on a rotor for 10 minutes. After pelleting

the samples at 2000 g for 5 minutes, the supernatants were removed and filtered

through glass wool. The silica pellet re - extracted a further 2 times before being dried

under a stream of N2.

40


Figure 2:2 - Representative 1D TLC of crude free polar mycobacterial lipids Plate was run in 14 : 6 : 0.8 CHCl3 : CH3OH : H2O, stained with 0.01 % 1, 6 - diphenyl - 1, 3, 5 - hexatriene in 9 : 1

petroleum ether : acetone. Plate visualised under long wavelength (366 nm) UV light.

Finely suspended and colloidal silica was removed from the samples by dissolving in 6 ml

of a 10 : 10 : 3 mixture of CHCl3 : CH3OH : H2O. Samples were mixed on a rotor for 30

minutes before the addition of 2.625 ml of CHCl3 and 1.125ml of H2O for a further 10

minutes. Separation of the phases was performed by centrifugation at 2000 g for 5

minutes and the upper, aqueous, phase and silica interface was removed. The remaining

lower organic phase was treated with 3 ml of a 3 : 47 : 48 mixture of CHCl3 : CH3OH : H2O

and mixed on a rotor for a further 10 minutes before centrifugation and removal of the

silica interface and upper layer. This process was repeated a further 3 times.

Finally, the silica free lower phase was dried under a stream of N2, resuspended in 5 ml of

a 2 : 1 mixture of CHCl3 : CH3OH and transferred to a clean, pre - weighed tube for

weighing and suspension.

41


Uninfected Cattle

Cattle between the ages of 6 and 36 months were obtained from herds within 4 -

yearly testing parishes with no history of a BTB breakdown in the past 4 years as

previously described(253). These animals were purchased when around 6 months old and

transported to AHVLA. Whilst at AHVLA they tested negative in both Bovigam IFNγ assay

and the SICCT. Further, they were also tested in the IFNγ test with the specific antigens

ESAT - 6 and culture filtrate protein - 10 kDa (CFP - 10) used as a combined peptide

cocktail and results confirmed the animals as negative.

M. bovis Infected Cattle

Cattle between the ages of 6 and 36 months were obtained from herds within 1 -

yearly testing parishes with a confirmed BTB breakdown within the past year as

previously described(253). These animals were purchased at the disclosing skin test and

transported to AHVLA. Whilst at AHVLA they regularly tested positive in both Bovigam

IFNγ assay and the SICCT. Further, they were also tested in the IFNγ test with the specific

antigens ESAT - 6 and CFP - 10 used as a combined peptide cocktail and results confirmed

the animals as M. bovis infected.

42


Isolation of Bovine PBMC from Whole Blood

The isolation of PBMC has been previously described(253). Briefly, whole blood was

mixed in equal amounts with sterile HBSS containing 10 U ml -1 heparin. This mixture was

overlaid onto Histopaque 1077 (Sigma) and centrifuged at 800 g for 40 minutes. The

PBMC interface was removed using a pastette and washed twice in HBSS containing

heparin. Cells were identified via trypan blue exclusion and enumerated using a

haemocytometer.

Isolation of CD14+ Monocytes from Bovine PBMC

Isolation of CD14+ cells was performed according to the manufacturer’s

instructions. PBMC were counted and suspended in 80 µl of MACS rinsing buffer (sterile

PBS containing 2mM EDTA and 035 % BSA or FCS) per 107 cells before the addition of 10

µl of MACS anti - CD14 MicroBeads (Miltenyi) per 107 cells. After a 15 minute incubation

at +4 oC on a rotator, cells were pelleted and resuspended in 500 µl per 108 cells and

passed through MACS LS columns as per the manufacturer’s instructions. The CD14+

fraction was counted and cells diluted to 1.5 x106 ml -1 in complete RPMI 1640.

Generation of Bovine Cultured Monocytes and MDDC

CD14+ monocytes were plated in 1 ml volumes of complete RPMI 1640 at 1.5 x106

ml-1 in 24 well plates (Nunc Nunclon) before adding either 1000 U ml -1 equine GM - CSF

43


(Kingfisher Biotech; RP0022E - 005) (cultured monocytes - CM) or 1000 U ml -1 equine GM

- CSF and 4 ng ml -1 bovine IL - 4 (AbD Serotec; PBP006) (monocyte derived DC - MDDC).

Cells were incubated at 37 oC + 5 % CO2 for 3 days.

After 3 days cells were harvested using a cell scraper and enumerated before being

plated out at 1.5 x106 ml -1 in fresh complete RPMI and lipid solution was added to the

wells. Cells were incubated for a further 12 - 16 hours before supernatants were

collected and cells harvested for subsequent flow cytometric analysis.

Measurement of IFNγ by BovigamTM ELISA

Levels of IFNγ were determined using the BovigamTM ELISA kit (Prionics AG,

Switzerland) in accordance with the manufacturer’s instructions. For the assessment of

adaptive immune responses to individual PIM molecules (see Chapter Six) responses

were considered positive if the OD450 exceeded the mean + 2 times the standard

deviation of OD450 for nil antigen stimulated cultures from all 10 animals.

Multiplex Measurement of Cytokine Production

Fresh and cultured monocytes and MDDC were derived as described above. After

3 days in culture, cells were stimulated with 20 µg ml -1 of either the polar or apolar lipid

fraction dissolved in RPMI as previously described. After 12 - 16 hours of stimulation,

supernatants were harvested and assayed for cytokine levels using the MSD multiplex

44


platform (MSD Systems, Washington) as described previously(254, 255). Briefly,

supernatants generated were analysed using a custom multiplex

electrochemiluminescent system which allows simultaneous detection of IL - 1β, IL - 6, IL -

10, IL - 12, MIP - 1β and TNFα (Meso Scale Discovery, Maryland, USA). Multiplex 96 well

plates were supplied with target capture antibodies spotted onto 6 separate carbon

electrodes in each well (commercially available antibodies: TNFα (Endogen, Rockford, IL,

USA); IL - 10 and IL - 12 (AbD-Serotec); IL - 1β, IL - 6 and human cross-reactive MIP - 1β

(MSD)).

Plates were blocked with MSD assay buffer for 30 minutes at room temperature before

the addition of samples or standards for 1 hour at room temperature. Standards were

prepared by serial dilution.

After incubation, plates were washed and combined biotinylated secondary detector

antibodies were added for a further hour. Finally, plates were washed, loaded with MSD

read buffer T and analysed using an MSD Sector Imager 6000.

Innate Cell Labelling & Analysis by Flow Cytometry

Cells were harvested from the well and labelled with the live / dead indicator

ViViD (Invitrogen) in PBS. Cells were then plated at approximately 50,000 stain-1 and

washed using 150 µl MACS rinse buffer before being stained for 15 minutes using either

anti - bovine CD14 (ccG33; Institute for Animal Health; 1 : 50), anti - equine MHCII

(MCA1085; AbD Serotec; 1 : 50), anti - bovine CD40 (IL - A156; cell supernatant, AHVLA; 1

45


: 10), anti - bovine CD80 (IL - A159; cell supernatant, AHVLA; 1 : 10), anti - bovine CD86 (IL

- A190; cell supernatant, AHVLA; 1 : 10), anti - bovine CD1b (CC14; AbD Serotec

MCA831G; 1 : 10) or an IgG1 isotype control (Av20; Institute for Animal Health; 1 : 50).

Labelled cells were washed using 150 µl MACS rinse buffer and secondary labelling was

performed using a 1 : 400 dilution of anti - IgG1 conjugated to R - Phycoerythrin (R - PE)

(Invitrogen; P21129) in 50 µl volumes for 10 minutes. After incubation, cells were

washed by the addition of 150µl PBS, pelleted and resuspended in 100 µl of 2 %

paraformaldehyde (Cytofix; BD Biosciences) for at least 30 minutes at 4 oC before analysis

on a CyAn ADP analyser.

For capture and analysis, initial gating was on the ViViDlo (live) cells into a subsequent

small cell / lymphocyte exclusion gate. An example of the gating strategy is shown in

figure 2:3. Normalisation of the median fluorescence intensity (MFI) was performed by

subtraction of the MFI of the Av20 isotype control from the MFI of the specific antibody

labelling (Δ MFI).

46


Figure 2:3 - Example of gating strategy for analysis of monocytes, MDM and MDDC A singlet gate (FSC lin v FSC area) removes debris and clusters of cells; ViViD staining allows isolation of live / viable

cells; a FSC vs SSC plot previously reverse gated on PBMC acts as a lymphocyte exclusion gate. Typical CD1b labelled MDDC are shown with their concurrent isotypte labelling.

Mixed Lymphocyte Reaction

Bovine MDDC and cultured monocytes were prepared as described above.

Following 3 days in culture, cells were pulsed with lipid antigen overnight before being

enumerated, washed to remove any cytokines and lipid from the media and incubated at

107 cells ml-1 in the presence of Mitomycin C at 100 μg ml-1 for 30 minutes at 37 oC + 5 %

CO2. Lipid pulsed, Mitomycin C treated MDDC or cultured monocytes were cultured in 1

ml at 37 oC + 5 % CO2 at 2 x105 with 1 x105 PBMC isolated from a second, allogeneic

animal. After 5 days, cells were pulsed overnight with 1 µCi well-1 of 3H-thymidine before

47


being harvested using a Harvester 96 Mach III (TomTec Inc, Hamden, CT, USA) as

described previously(256). Lymphocyte proliferation was assessed by the increased cellular

incorporation of 3H-thymidine which was measured using a MicroBeta2 2450 (Perkin

Elmer, Waltham, MA, USA).

Lymphocyte Transformation Assay

PBMC were isolated from heparinised whole blood using Histopaque 1077 (Sigma)

gradient centrifugation (as described above) and 2 x105 cells well -1 plated out in triplicate

wells after resuspension in complete RPMI 1640. Cells were mixed with lipid antigen,

complete RPMI as a negative control, pokeweed mitogen (PWM) at 10 µg ml -1 as a

positive control, or a 1 : 100 dilution of PPD - B.

Measurement of proliferation by the incorporation of 3H - Thy has been previously

described(256). Briefly, after 4 days of incubation at 37 oC + 5% CO2, 30 µl of supernatant

was harvested for cytokine analysis and cells were pulsed with 1 µCi 3H - Thy well -1 in 30

µl to replace the removed supernatant. After a further 16 - 24 hours incubation the cells

were washed and harvested using a semi - automated harvester (Tomtec) and

incorporation of 3H - Thy was read using MicroBeta2 (Perkin Elmer).

For the assessment of adaptive immune responses to individual PIM molecules (see

Chapter Six) responses were considered positive if the counts per minute (CPM) exceeded

the mean + 2 times the standard deviation of the CPM for nil antigen stimulated cultures

from all 10 animals.

48


Lymphocyte Labelling & Analysis by Flow Cytometry

Bovine PBMC were isolated as described above and labelled with CellTrace Violet

(Invitrogen Molecular Probes, Paisley, UK) in accordance with the manufacturer’s

instructions. Briefly, PBMC were suspended at 1 x107 ml-1 in pre - warmed PBS and 5 mM

CellTrace Violet was added to a final working concentration of 1 μM. Cells were

incubated at 37 oC for 20 minutes before unbound dye was quenched with 5 times the

labelling volume of complete cell culture medium at 37 oC for 5 minutes. Finally, cells

were pelleted and washed in pre - warmed complete cell culture medium, plated at 2

x105 cells well-1 and incubated at 37 oC + 5 % CO2 for 5 days in the presence of antigen.

Cultured cells were harvested and resuspended in flow cytometry buffer (PBS containing

2 % FCS and 0.05 % NaN3) and labelled for 15 mins with near infrared live / dead viability

dye (NIRViD, Invitrogen Life Technologies, Paisley, UK) and mouse anti - bovine CD335

(AKS1, AbD Serotec, Oxfordshire, UK). Cells were washed in flow cytometry buffer and

secondary labelling of anti - CD335 was performed using a 1 : 400 dilution of rat anti -

mouse IgG2a conjugated to allophycocyanin for a further 15 minutes. After a subsequent

wash, cells were further labelled with combinations of R - PE Zenon labelled (Invitrogen

Life Technologies, Paisley, UK) mouse anti - bovine CD3 (MM1A; WSU Monoclonal

Antibody Centre, Pullman, Washington, USA), mouse anti - bovine CD4 conjugated to

alexafluor 647 (CC30, AbD Serotec, Oxfordshire, UK), mouse anti - bovine CD8 conjugated

to alexafluor 647 (CC63, AbD Serotec, Oxfordshire, UK) and alexafluor 488 Zenon labelled

(Invitrogen Life Technologies, Paisley, UK) mouse anti - bovine γδ TCR1 (GB21a; WSU

Monoclonal Antibody Centre, Pullman, Washington, USA). Finally, labelled cells were

washed in flow cytometry buffer and resuspended in 150 μL of 2 % paraformaldehyde

49


(Cytofix; BD Biosciences, Oxfordshire, UK) for at least 30 min at 4 °C before analysis on a

CyAn ADP analyser. For capture and analysis, initial gating was on single, NIRViDlo (live)

cells into a subsequent lymphocyte gate before gating on CellTrace Violetlo cells and

assessing surface phenotype (figure 2:4).

Figure 2:4 - Example of gating strategy for analysis of proliferative cells Flow cytometric gating strategy: single, live CD3+ CD335+ lymphocytes are assessed for CellTrace Violet labelling and CellTracelo cells gated for phenotyping. Numbers represent the percentage of proliferating cells in response to each

antigen.

Monoclonal Antibody Blocking of MHCII and CD1

Bovine PBMC were isolated as described above and incubated for 2 hours in the

presence of either anti - equine MHCII (MCA1085; AbD Serotec), anti - ovine CD1

(MCA2212; AbD Serotec), anti - galline Bu - 1a/b as an isotype control (MCA5764; AbD

Serotec) or no monoclonal antibody. All antibodies were sterile and contained no

50


preservatives. After 2 hours, antigens were added and proliferation measured as

described for the Lymphocyte Transformation Assay

Removal of Lipopeptide by Proteinase Treatment

The use of Proteinase K to remove lipopeptide has been previously described(257).

For treatment, 20 µg of lipid was combined with 5 µg of Proteinase K from Tritirachium

album (Sigma Alrdich; P6556) in PBS in a PCR plate. Using a thermocycler, the mixture

was incubated at 50 oC for 30 minutes and the Proteinase subsequently inactivated by

increasing the temperature to 80 oC for a further 30 minutes. After inactivation, the

samples were chilled to 4 oC. Mock treatment was performed by incubating the 5 µg of

Proteinase K at 80 oC for 30 minutes before adding 20 µg of lipids and continuing the

treatment as above. Proteinase K treated and mock treated lipid antigens were

suspended as described above and used to stimulate PBMC as described for the

Lymphocyte Transformation Assay.

Data & Statistical Analysis

All data representation and statistical analysis was performed using GraphPad

Prism version 5 and GraphPad InStat version 3. Densitometry analysis of TLCs was

performed using the gradient selection tools in Quantity One version 4.6.9. All flow

cytometric data was captured using a 9 colour Beckman Coulter CyAn ADP analyser and

data was captured and analysed using Summit v4.3.

51

Chapter Three Preparation & Characterisation of Crude Lipid Extracts

Chapter Three

Preparation & Characterisation of Crude Lipid Extracts

Background

To enable analysis of specific M. bovis - derived lipids, and subsequently the host

response to them, lipids first need to be extracted from bacterial cells. Furthermore, the

content of these extracts must be analysed to show the presence of lipids and allow for

basic identification of specific lipid moieties.

The extraction of bacterial lipids was first described in 1959 by Bligh and Dyer(258) and was

further pioneered by Minnikin(128, 259, 260). Extraction of free lipids essentially removes the

outer - membrane or leaflet of the mycobacterial cell wall, leaving the long chain

molecules, such as the mycolic acids, intact in the defatted cells(259, 260). The process

(illustrated in figure 2:1) involves treatment of freeze dried bacterial cells with a mixture

of methanol, saline and petroleum ether. From this mixture, the cells and aqueous phase

are removed using chloroform and methanol leaving the extractable free apolar lipids.

Further treatment of the secondary extract with chloroform and saline yields the

remaining polar lipids.

52


Primary analysis of these lipid extracts is usually performed using 5 specially developed

thin - layer chromatography (TLC) systems (as detailed in table 2:1). Four of these

systems (A - D) are used to analyse the apolar lipids, while the polar fraction was analysed

with using System D and E.

These systems cover the polarity range of the extracted lipids; system A is used to detect

triacyl glycerols (TAG), menaquinones (MQ) and the phthiocerol family of mycocerosic

acids (phthiocerol dimycocerosates; PDIMs)(261). Identification of unesterified fatty acids

and acyltrehaloses is performed using system B, the second least polar system(252). The

third, slightly more polar, system C enables detection of more fatty acids and some

glycosides, which are structurally related to the PDIMs. System D, the most polar system

used in the analysis of the non - aqueous petroleum ether extracts, resolves the trehalose

based mycolates (such as trehalose dimycolate [TDM] or cord factor), the acylated

trehaloses such as diacyl trehalose (DAT) and the sulpholipids (such as SL - 1).

As well as the petroleum ether extracts, system D is also used to analyse the least polar

entities in the chloroform : methanol extracts and enables identification of the

glycopeptidolipids and small amounts of sulpholipids. The most polar TLC system is only

useful in the analysis of the chloroform : methanol extracts and was introduced to the

standard array of systems in 1966(262). The system primarily resolves glycolipids and

phosphatide based lipids such as diphosphatidylglycerol (DPG),

phosphatidylethanolamine (PE) and phosphatidylinositol (PI) and its mannosides (PIMs).

53


TLC plates may be stained to further aid identification of individual lipid classes and

functional residues. Treatment with MPA, usually dissolved in ethanol or another polar

solvent, stains phenolics, hydrocarbon based waxes and alkaloids. Used as a general

stain, it allows identification of any lipid molecules which appear brown after staining and

subsequent heating. Upon charring, reduction of MPA (Mo6+) to Mo5+ and Mo4+

compounds by organic compounds causes the production of dark spots against a light

green background. Alternatively, plates may be sprayed with a solution of ninhydrin

dissolved in n - butanol. Originally discovered accidentally by Siegfried Ruhemann in

1910(263) who observed the ability of the compound to react with amine residues forming

a purple coloured product(264), ninhydrin was originally used to detect trace amounts of

amino acids in biological samples(265, 266) and has been used to detect amino acids in TLC

since the 1950s(267) and has been used in the detection of fingerprints since 1959(268). The

formation of either Ruhemann’s Purple upon reaction of ninhydrin with a primary amine

or a light yellow compound upon reaction with a secondary amine allows for the

identification of lipopeptides co - extracted with the lipid fractions.

While initial experiments were performed using AF 2122/97 - derived lipids (see Chapter

Four), biosafety concerns over the high cell mass required for large scale lipid extraction

meant that an alternative source of high volume mycobacterial culture was required. To

this end, pellicle grown M. bovis AN5 was sourced and used for subfractionation work

(see Chapter Five). This chapter documents the extraction of lipids from both the

reference strain of M. bovis, AF 2122/97 and the PPD - B production strain, AN5 and the

identification of the individual lipid components by 2D TLC.

54


This chapter addresses the hypothesis that lipids from the virulent M. bovis strains AF

2122/97 and AN5 could be extracted based on their solubility in aqueous and non -

aqueous solvents. These fractions could be subsequently characterised, quantified and

their main components identified using previously published analysis of mycobacterial

lipids(252).

55


Results

Extraction & Analysis of Lipids from M. bovis AF 2122/97

Lipids were extracted from the reference strain of M. bovis (AF 2122/97) using the

methods described above and were stained using both MPA and ninhydrin. AF 2122/97 -

derived lipids are shown in figure 3:1 (MPA stained) and figure 3:2 (ninhydrin stained).

Analysis of the petroleum ether extract (containing the apolar fraction) with the least

polar solvent system (figure 3:1 A) enabled the identification of TAG, MQ and the PDIMs.

System B (figure 3:1 B) resolved pentacyl trehalose (PAT) and the co - located

monomycolyl glycerol (MMG) and phenolic glycolipid (PGL), a lipid unique to M. bovis.

The use of solvent system C (figure 3:1 C) allowed further discrimination of MMG and PGL

whilst the most polar constituents of the petroleum ether extract, the cord factors TDM

and TMM and glucose monomycolate (GMM) were seen using system D (figure 3:1 D).

56


Figure 3:1 - 2D TLC analysis of crude, free lipids extracted from M. bovis AF 2122/97 and stained with MPA (A - D): Apolar fraction analysed with TLC systems A, B, C and D. (E and F): Polar fraction analysed with systems D and E.

57


Analysis of the chloroform : methanol extract (which contains the polar lipid fraction)

with system D (figure 3:1 E) resolved TDM and TMM. More individual lipids were seen

using the most polar system (figure 3:1 F) which mainly resolved a variety of PIMs and

miscellaneous phospholipids.

Staining of plates with ninhydrin allows for the discrimination of amines and amino acids

and is commonly used to assess the presence of lipopeptide in lipid preparations. As can

be seen in figure 3:2 A - D, no ninhydrin staining can been seen in the petroleum ether

fraction.

Analysis of the polar, chloroform : methanol extract, stained with ninhydrin showed no

lipopeptide present in TLC system D (figure 3:2 E). In contrast, lipopeptide was resolved

using system E (figure 3:2 F). A primary amine containing molecule was found co -

located with the dimannoslylated PIMs (figure 3:2 F, red arrow) and an unknown

secondary amine containing molecule was seen to have migrated near to the PE spot

(figure 3:2 F, yellow arrow).

58


Figure 3:2 - 2D TLC analysis of crude, free lipids extracted from M. bovis AF 2122/97 and stained with ninhydrin (A - D): Apolar fraction analysed with TLC systems A, B, C and D. (E and F): Polar fraction analysed with systems D and E.

Red arrows indicate primary amines; yellow arrows indicate secondary amines; black arrows indicate charred lipid for reference. Identifiable spots are named.

59


Extraction & Analysis of Lipids from M. bovis AN5

To perform larger scale lipid extraction for subsequent subfractionation, a far

greater bacterial cell mass was required than had been used thus far (see Chapter Five).

From a biosafety perspective, culturing such large volumes of M. bovis AF 2122/97 was

considered unreasonable as high yield, pellicle grown M. bovis AN5 was already available

for use. M. bovis AN5 - derived crude lipid extracts were analysed using the same solvent

systems (table 2:1) and stained with the same reagents (MPA and ninhydrin) as shown for

AF 2122/97 lipids.

As was seen for AF 2122/97 - derived lipids, the apolar fraction, resolved using system A,

contained TAG, MQ and the PDIMs (figure 3:3 A). PAT, MMG and PGL were identified

using system B (figure 3:3 B) and MMG and PGL further separated with the use of system

C (figure 3:3 C). TMM, TDM and GMM were again resolved using system D (figure 3:3 D).

60


Figure 3:3 - 2D TLC analysis of crude, free lipids extracted from M. bovis AN5 and stained with MPA (A - D): Apolar fraction analysed with TLC systems A, B, C and D. (E and F): Polar fraction analysed with systems D and E.

61


Similarly, the polar fraction from M. bovis AN5 contained TMM and TDM (figure 3:3 E)

and the same array of PIMs, phospholipids, PE and PI (figure 3:3 F) as the M. bovis AF

2122/97 - derived lipids (figure 3:1 E - F).

As was seen with AF 2122/97 - derived lipids, ninhydrin staining of the M. bovis AN5 -

derived lipid fractions showed no lipopeptide present in the apolar fraction (figure 3:4 A -

D) and no ninhydrin staining was seen in the polar fraction separated with system D

(figure 3:4 E).

Finally, ninhydrin staining of system E revealed amine containing molecules co- locating

with the dimannoside PIMs (figure 3:4 F, red arrow) and an unknown secondary amine

located close to the PE spot (figure 3:4 F, yellow arrow).

62


Figure 3:4 - 2D TLC analysis of crude, free lipids extracted from M. bovis AN5 and stained with ninhydrin (A - D): Apolar fraction analysed with TLC systems A, B, C and D. (E and F): Polar fraction analysed with systems D and E.

Red arrows indicate primary amines; yellow arrows indicate secondary amines; black arrows indicate charred lipid for reference. Identifiable spots are named.

63


Abundance Analysis of the Crude Lipid Fractions

The use of densitometry analytical techniques allows for the quantification of

individual lipids on the TLC plates and thus enables calculation of the relative abundance

of each lipid. As the same quantity of each crude lipid fraction (100 µg) was loaded onto

each TLC plate, it is possible to directly compare all of the plates containing lipids from

either the polar or apolar fractions. Of note, differences in the separation of the spots

made the selection of individual spots challenging which introduced a degree of

variability into the quantification.

Using false coloured TLC images of the AF 2122/97 - derived polar fraction (figure 3:5 A -

D) the abundance of the individual lipids is shown in table 3:1.

In total, around 16 % of the apolar lipid fraction can be identified by this method. The

largest proportion of identifiable lipid is made up of TAG (4.77 % of the apolar fraction)

followed by the PDIMs (3.81 %). In system B (figure 3:5 B) PGL and MMG co - locate on

the plate and cannot be resolved individually, however in system C (figure 3:5 C) they can

be resolved separately. MMG makes up the larger proportion of these lipids (2.15 % of

the total apolar fraction) while PGL accounts for 1.2 % of the total lipid preparation.

GMM, visible in system D (figure 3:5 D) makes up 1.62 % of the total fraction and the

remaining lipids all account for less than 1 % of the fraction each (MQ: 0.89 %, PAT: 0.79

%, TDM: 0.72 % and TMM: 0.60 %).

64


Figure 3:5 - False coloured densitometry analysis of lipids extracted from M. bovis AF 2122/97 and analysed by 2D TLC (A - D): Apolar fraction analysed with TLC systems A, B, C and D. (E and F): Polar fraction analysed with systems D and E.

65


As shown in table 3:2, within the AF 2122/97 - derived polar lipid fraction visible in

systems D and E (figure 3:5 E and F) TDM is the most abundant identifiable lipid species

(4.04 %) with TMM second comprising 1.22 % of the fraction. The lipids resolved by

system E (figure 3:5 F) make up less than 1 % of the total polar fraction each, however in

total these 8 lipid moieties account for 4.09 % of the fraction.

Analysis of the AN5 - derived polar fraction (figure 3:6 A - D) was also performed and the

abundance of the individual lipids is shown in table 3:1.

In total, around 9 % of the apolar lipid fraction can be identified by this method. The

largest proportion of identifiable lipid is made up of the PDIMs (3.36 % of the apolar

fraction) followed by MMG (1.36 %), TAG (1.1 %) and MQ (0.94 %). The remaining 2 % of

identifiable lipids consisted of PAT (0.58 %), PGL (0.5 %), TDM (0.43 %), GMM (0.37 %)

and TMM (0.15 %).

In total, only 4.5 % of the AN5 - derived polar fraction could be identified (figure 3:6 E and

F; table 3:2). The largest component was Ac1PIM6 (1.59 %) followed by TDM (0.93 %).

Ac2PIM6 and DPG were present in similar quantities (0.46 %) and the remaining 1 % was

accounted for by P (0.28 %), Ac2PIM2 (0.26 %),TMM (0.21 %), PE (0.16 %), Ac1PIM2 (0.08

%) and PI (0.07 %).

66


Figure 3:6 - False coloured densitometry analysis of lipids extracted from M. bovis AN5 and analysed by 2D TLC

(A - D): Apolar fraction analysed with TLC systems A, B, C and D. (E and F): Polar fraction analysed with systems D and E.

67


Table 3:1 - Densitometry of the apolar lipid fractions. Volumes (intensity x area) are used to calculate to amount of total identifiable lipid within the apolar fractions. *Lipids are co - located and inseparable in this system and have not been used as part of the total calculation.

Solvent System

Identified lipids

% Total lipid AF 2122/97

% total lipid AN5

A TAG MQ PDIMs

4.77 0.89 3.81

1.10 0.94 3.36

B PGL / MMG PAT

3.44* 0.79

0.93* 0.58

C MMG PGL

2.15 1.20

1.36 0.50

D TMM TDM GMM

0.60 0.72 1.62

0.15 0.43 0.37

Total identifiable lipids 16.54 % 8.79 %

Table 3:2 - Densitometry of the polar lipid fractions. Volumes (intensity x area) are used to calculate to amount of total identifiable lipid within the polar fractions.

Solvent System

Identified lipids

% Total lipid AF 2122/97

% total lipid AN5

D TMM TDM

1.22 4.04

0.21 0.93

E Ac1PIM6 Ac2PIM6 Ac1PIM2 Ac2PIM2 PI P DPG PE

0.28 0.53 0.62 0.43 0.69 0.37 0.52 0.66

1.59 0.46 0.08 0.26 0.07 0.28 0.46 0.16

Total identifiable lipids 9.35 % 4.50 %

68


Using these methods it was possible to identify approximately 25 % of the total lipids

extracted from M. bovis AF 2122/97 and approximately 13 % of those from M. bovis AN5.

Whilst identical compounds could be identified qualitatively in the lipid profiles of both

strains, there were obvious quantitative differences. Thse will be discussed in the

following section.

69


Discussion

Extraction of mycobacterial lipids and their subsequent analysis by 2D TLC has

been previously described for M. tuberculosis (269), but little data exists on total lipid

profiling of M. bovis. Previous work by Dandapat et al.(270) attempted to characterise M.

bovis AN5 based on the expression of PGL and PDIMs, but only as a tool for identification

of the organism. Further, the use of M. bovis AN5 may not have been representative of

the AF 2122/97 reference strain. Presented here is the first biphasic extraction and

complete analysis of the lipids of M. bovis AF 2122/97.

In figure 3:1, the use of MPA staining allowed the identification of a broad range of

characteristic mycobacterial lipids including PDIMS (figure 3:1 A), the M. bovis

characteristic PGL(271) (figure 3:1 B and C), TDM (figure 3:1 D and E) and PIMs (figure 3:1

F). As expected, no sulphoglycolipid was found (figure 3:1 D)(210, 211). Interestingly, TDM

was found in both the polar and apolar extracts (figure 3:1 D and E). This may be related

to its particularly amphipathic nature(272) and variable acylation states which are known

to alter its hydrophobicity(273, 274) and may cause the molecule to split differentially across

the biphase interface during lipid extraction.

No systematic lipopeptide profiles have been published for any mycobacterial species,

despite the fact that lipopeptides from a variety of mycobacterial species have been

shown to modulate immune responses(275-277) and that some responses attributed to

lipids may be driven by lipopeptide instead(257).

70


Analysis of the AF 2122/97 - derived lipid fractions by ninhydrin indicated no lipopeptide

presence in the apolar fraction (figure 3:2 A - D) but both primary and secondary amine

containing molecules in the polar lipid fraction (figure 3:2 F). Interestingly, one primary

lipopeptide co - locates precisely with PI suggesting that the amine containing molecule

may be physically associated with the lipid. A secondary amine was seen which located

near the PE spot (figure 3:2 F). The identity of both these molecules is unknown, as is the

reason for their co - location with the lipid molecules although it may be that any

lipopeptide present is chemically bonded with the lipids.

Analysis of the M. bovis AN5 - derived lipids with the standard TLC systems and stained

with MPA (figure 3:3) allowed for a comparison between the 2 bacterial strains. Overall,

the same lipid molecules were present in the apolar (figure 3:3 A - D) and polar (figure 3:3

E - F) lipid fractions. However, it is clear that the lipids derived from M. bovis AN5 do not

separate in exactly the same manner as those from AF 2122/97 and form spots of

different shapes. From the analysis performed here it is not possible to tell if these

differences are due to the synthesis of inherently different forms of these lipids in the 2

strains, or if the differing conditions of bacterial culture have affected lipid synthesis; a

factor known to play a role in the lipid production of mycobacteria(278, 279).

Lipids from M. bovis AN5 were also stained with ninhydrin (figure 3:4). Although not as

clear as for AF 2122/97 - derived lipids, the primary amine staining co - located with PI is

present in AN5 - derived lipids, as is the secondary amine seen located near the PE spot

(figure 3:4 F).

71


It is worth noting that the lipids from M. bovis AN5 separated less cleanly than those from

AF 2122/97. This is most clearly demonstrated in the most polar solvent systems used to

analyse the apolar lipids (figure 3:3 C - D) where lipids dissolved in the mobile phase have

left streaks or smears in both directions of the TLC. Such smearing or streaking is often

due to overloading of the analyte on the TLC plates, however this is unlikely in this

instance. Each plate shown in figure 3:1, figure 3:2 and figure 3:4 has 100 µg of lipid

loaded at the origin and spots are consistently located and well defined. Perhaps more

likely is that damage was caused to some of the AN5 - derived lipid molecules by

autoclaving at 134 oC for 1 hour. For technical reasons, it was not possible to heat kill the

pellicle grown M. bovis AN5 by the same method as the AF 2122/97, which was heated to

between 80 oC and 90 oC for 1 - 2 hours, and this extra heat may have denatured some of

the lipid structures causing them to dissolve into or precipitate out of the mobile phase

less efficiently.

The effect of growth as a pellicle upon lipid composition is not well understood, despite

the fact that growth at the interface between medium and air plays an important role in

many other bacterial genera. Vibrio cholerae, some streptococci and staphylococci are

known to form biofilms which have been implicated in their virulence(280-283). It has been

suggested that M. tuberculosis is capable of forming biofilms and some evidence has

been found that biofilm - like structures form in guinea pig lung lesions which enhance

antimicrobial resistance and virulence of the bacteria(284). However little information

exists on changes in lipid profile, synthesis or metabolism when mycobacteria are

cultured in their planktonic form. It has been shown that the use of carbon limiting

chemostats to replicate bacterial growth rates of active infection and a slower dormant

72


phenotype led to significant changes in total lipid composition of BCG(285). However, this

study used a single lipid extraction and subsequent elemental analysis by combustion to

ascertain the authors’ findings. Other work has demonstrated an alteration of the

transcriptional response which led to the identification of a novel wax ester when M.

tuberculosis was grown in iron limited conditions(278). It is also known that keto - mycolic

acids are essential for pellicle formation by M. tuberculosis(286) and that pellicle formation

can allow drug resistant bacteria to replicate(287).

Overall, the lipid profiles obtained by extraction of the apolar and polar fractions from

both AF 2122/97 and AN5 are remarkably similar. All lipids migrated to the same

locations upon 2D TLC analysis and all lipids of known identities were present in both

strains.

In an attempt to further characterise the lipid fractions, densitometry analysis was

performed to allow relative quantification of the lipids present in the polar and apolar

subfractions. This technique is more commonly used for imaging gels analysed using a gel

documentation system, however it was possible to load in high resolution images of the

TLC plates, false colour them and select individual spots for analysis (figure 3:5 and figure

3:6).

Using the images of the AF 2122/97 - derived lipids, each spot of a known identity was

selected, along with the entire plate. As the plates shown in figure 3:5 A - D all contain

100 µg of the apolar lipid fraction it is possible to collate the information for the total

lipid on each plate and calculate the volume of each individual spot. In total, the

73


identifiable apolar lipids from AF 2122/97 constitute just over 16 % of the total, MPA

stained lipid in the fraction (table 3:1). Similarly, just over 9 % of the polar lipids could be

identified (table 3:2).

Interestingly, a smaller proportion of the AN5 - derived lipid fractions could be quantified

despite the presence of the same lipid molecules. As discussed above, growth conditions

could have played a significant role in the different relative abundances of the lipid

molecules. It is also likely that autoclave treatment of the AN5 pellicle, and the

subsequent damage to the lipid molecules, affected the ability to perform the

densitometry analysis. More spreading and smearing of lipid was present in the AN5 -

derived lipids upon TLC analysis which compromised the ability of the analysis software

to select the edges of the spots. Therefore it is possible that more identifiable lipid is

present than can be reported here.

These data demonstrate that considerably more lipid is present in these fractions than

can be identified with these TLC systems. Further evidence of this can be seen in the

solvent front and origins of the TLC plates. The least polar solvent system used (system

A) allows only the least polar lipids to dissolve into the mobile phase and migrate over the

plate. However, some extremely apolar lipid material is contained within the solvent

front and can be seen in the apolar lipids from both AF 2122/97 (figure 3:1 A) and AN5

(figure 3:3 A).

Similarly, system E is the most polar solvent system currently in use and allows the most

polar lipids to dissolve. However MPA stained lipid material is still present at the origin

74


(figure 3:1 F and figure 3:3 F) suggesting that lipid, or lipid containing, material of greater

polarity is present in the fraction which requires further analysis in more polar solvents.

In conclusion, this chapter demonstrates the ability to chemically extract aqueous and

non - aqueous lipid fractions from virulent strains of M. bovis. Further, it was possible to

characterise these fractions by MPA stained 2D TLC and identify the main lipid families

contained within the fractions of both strains of M. bovis. Ninhydrin staining of these

TLCs also enabled the discovery of lipopeptides present in the polar fraction from both

strains. Finally, the application of densitometrical analysis allowed the quantification of

the lipid species within the fractions. Having generated and characterised these

fractions, both qualitatively and quantitatively, they could then be used to stimulate

bovine innate immune responses.

75


Chapter Summary

• Objective

The extraction of polar and apolar lipid fractions from M. bovis strains AF

2122/97 and AN5 and their subsequent characterisation by 2D TLC and

densitometry

• Results

Polar and apolar lipid fractions were successfully extracted from both M. bovis

strains. A broad range of lipids was identified in both polar and apolar

fractions and qualitative TLC analysis showed that AF 2122/97 and AN5

fractions consist of similar lipid species. Lipopeptide was identified in the polar

fractions from both strains and these were qualitatively similar.

Densitometrical analysis showed that identifiable lipids composed

approximately 25 % of the AF 2122/97 - derived fractions. The AN5 - derived

fractions were composed of 13 % identified lipids.

• Conclusions

Polar and apolar lipid fractions can be chemically extracted from virulent M.

bovis and have been characterised and quantified here for the first time.

Further, the polar fraction has been shown to contain lipopeptides. Much of

the content of the fractions is unknown and requires the development of new

analytical techniques as the current TLC systems will not resolve these

molecules.

76

Chapter Four Effects of Crude Lipids on Bovine Innate Immune Cells

Chapter Four

Effects of Crude Lipids on Bovine Innate Immune Cells

Background

It is widely accepted that the interaction between host and pathogen is critical in

establishing an appropriate and effective immune response which controls the invading

pathogen and protects the host from the effects of infection. The role of macrophages in

protection against tuberculous infection was discovered over 60 years ago(160-162). Much

work was performed in the 1980s which lead to a greater understanding of the

interaction between the macrophage and other host immune components including a

method of macrophage mediated bacterial killing using hydrogen peroxide(163) and the

critical role of IFNγ in the activation and enhancement of macrophages and their

killing(164, 165). Since then the interaction between mycobacterial species, usually M.

tuberculosis or BCG, and APCs has been heavily studied.

Ligation of different receptors drives different effector functions, some specifically

promoting phagocytosis (e.g. scavenger receptors) and others triggering non - phagocytic

maturation or activation (such as the TLRs). It is the selective and multiple ligation of

these receptors which dictates the effector function of the APC(167, 168). Further, it has

77


been reported that M. tuberculosis interaction with TLR2 mediates TNFα release(174).

Genetic removal of TLR2 in murine models leads to impaired immune responses to M.

avium(172) and recent work has shown impaired anti - mycobacterial activity of

macrophages and reduced TNFα production in TLR2 deficient mice(173). It has also been

shown that triacylated lipomannans bound to the TLR2 - TLR1 heterodimer induced the

production of IL - 12p40 and nitric oxide and that tetra - acylated lipomannans exerted

the same effect but through TLR4(171). In contrast, mannose receptor mediated

phagocytosis of M. tuberculosis has been shown to generate Th2 T cell responses

characterised by production of IL - 10, CCL22 and CCL17(178, 179) and ligation of the MR by

mannosylated LAM (ManLAM) inhibits IL - 12 production(180).

The ability to derive DCs and macrophages from blood monocytes in vitro and harness

their antigen presenting ability was first described by Sallusto & Lanzavecchia(288).

Previous to this, in vitro derivation of DCs required either cord blood or bone marrow

precursor cells and was performed using GM - CSF and TNFα(289). In their ground breaking

work, Sallusto & Lanzavecchia demonstrated not only the ability to culture DC from

peripheral blood monocytes, but that TNFα inhibits DC development and that IL - 4

enhanced it. This has since proven to be a major advancement, thanks to the simplicity

of their method, and has led to a huge increase in the study of DCs.

It is clear that innate recognition of mycobacteria is critical in generating an appropriate

immune response and that the mammalian immune system has many ways of sensing

mycobacteria. This chapter addresses the hypothesis that the lipid fractions generated

78


and characterised in Chapter Three would be capable of driving functional and

phenotypic responses in bovine antigen presenting cells.

79


Results

Characterisation of Cultured Monocytes and Monocyte Derived DC

CD14+ monocytes were isolated from uninfected cattle and cultured for 3 days in

the presence of either GM - CSF (cultured monocytes) or GM - CSF and IL - 4 (MDDC).

Cultured monocytes displayed little morphological change from the freshly isolated cells

(figure 4:1 A). However, after 3 days of culture in the presence of GM - CSF and IL - 4, the

cells displayed characteristic DC - like morphology primarily characterised by flattening

and the extension of dendritic processes (figure 4:1 B).

CD14+ cells were also cultured for 3 days without the addition of cytokines to the

medium. These cells did not display any flattening or process extension and appeared

almost morphologically indistinguishable to those cultured with GM - CSF (not shown).

The majority of the untreated cells did not survive 3 days of culture and, upon

enumeration using trypan blue to discriminate live cells, yields were typically very low

with only 10 - 15 % the cells viable. Further, upon subsequent flow cytometric analysis,

up to 95 % of these trypan blue counted cells were considered dead after staining with

ViViD, therefore these cells were not used in any further studies.

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Figure 4:1 - CD14+ cells after culture for 3 days in the presence of either GM - CSF or GM - CSF and IL - 4. (A) bovine GM - CSF; (B) bovine GM - CSF and IL - 4. Arrow (i) indicates flattening and enlarging of cells. Arrow (ii)

indicates a dendritic cellular processes. Magnification 400x using phase contrast microscopy.

81


Having shown morphologic differences between the culture conditions, the cells were

further characterised using flow cytometry to assess the expression of several key

molecules to define their individual phenotypes.

Figure 4:2 summarises the normalised expression of the surface molecules MHCII (A),

CD86 (B), CD40 (C), CD80 (D), and CD1b (E) on freshly isolated monocytes, GM - CSF

treated cells (regarded as cultured monocytes [CM]) and GM - CSF and IL - 4 treated cells

(regarded as MDDC). When comparing monocytes with CM, a trend was evident for

lower expression of MHCII on cultured monocytes (figure 4:2 A). This small reduction in

MHCII expression was accompanied by a similar trend for a reduction in CD86 expression.

No difference was seen in the levels of CD40 expressed by either monocytes or CM

(figure 4:2 C). CM expressed significantly more CD80 than monocytes expression when

compared to monocytes (figure 4:2 D). Finally, neither monocytes nor CM expressed any

CD1b (figure 4:2 E).

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Figure 4:2 - Phenotype of fresh CD14+ monocytes, cultured monocytes (CM) and cultured DC (MDDC) Median fluorescence intensity of (A) MHCII, (B) CD86, (C) CD40, (D) CD80, (E) CD1b. Points represent individual

animals; bar represents mean Δ MFI. * p < 0.05, ** p < 0.01 using repeated measures ANOVA with Bonferroni multiple comparisons test.

Comparison of MDDC with monocytes showed that there was a trend for increased levels

of MHCII expression on MDDC (figure 4:2 A). No difference was seen in the levels of

CD86 expressed by either MDDC or monocytes (figure 4:2 B) or CD80 (figure 4:2 D).

However, MDDC were found to express significantly higher levels of CD40 than

83


monocytes (figure 4:2 C) and, perhaps most strikingly, MDDC were the only cells to

express the lipid - specific antigen presentation molecule CD1b (figure 4:2 E).

Finally, comparison of CM with MDDC showed a strong trend for higher levels of MHCII

expression on MDDC (figure 4:2 A) and similar levels of CD86 expression on both cell

types (figure 4:2 B). MDDC expression of CD40 was significantly higher than that seen on

CM (figure 4:2 C) while CM expression of CD80 was significantly higher than that by

MDDC (figure 4:2 D). As was seen with monocytes, MDDC expressed significantly

increased levels of CD1b in comparison to CM, which expressed no CD1b (figure 4:2 E).

Overall, clear differences were apparent between bovine monocytes, cultured monocytes

and MDDC. Cultured monocytes were characterised by their decreased expression of

MHCII (Δ MFI = 114 compared to Δ MFI = 172 on monocytes) and CD86 (Δ MFI = 35

compared to Δ MFI = 50 on monocytes) combined with the subsequent significant

increase in CD80 expression (Δ MFI = 23 compared to Δ MFI = 10 on monocytes; p <

0.0005).

MDDC were defined by their significantly higher levels of CD40 (Δ MFI = 126 compared to

Δ MFI = 41 on monocytes; p < 0.001) and the presence of CD1b (Δ MFI = 30 compared to

Δ MFI = 0 on monocytes and MDM). Furthermore, MDDC displayed a characteristic

flattened morphology and the extension of long cellular process.

84


Cytokine Responses to Crude Mycobacterial Lipids

In order to assess the effect of M. bovis - derived lipids on these bovine innate

cells, cytokine production was measured after overnight stimulation with the lipid

fractions (figure 4:3).

Significantly increased IL - 10 secretion was seen from all 3 cell types following

stimulation with the polar lipid fraction (figure 4:3 A). Strong IL - 10 responses were seen

for 3 animals, while only modest increases were noted for the remaining cattle (figure 4:3

A). In contrast, little or no significant increase in IL - 10 production was seen following

stimulation with the apolar lipid fraction, although apolar lipids induced some IL - 10

production by cultured monocytes and MDDC from 3 animals. All responses to apolar

lipids were by far lower than those induced by the polar lipid fraction

IL - 12 levels in the culture supernatants were measured simultaneously and the results

are shown in figure 4:3 B. Stimulation with the polar lipid fraction induced a large

increase in IL - 12 production by MDDC, while lower responses were also observed from

monocyte and cultured monocyte populations. In contrast to the polar lipids, stimulation

with the apolar lipid fraction resulted in minimal increases in IL - 12 production by

monocytes, cultured monocytes or MDDC.

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Figure 4:3 - Effect of stimulation with the polar and apolar lipid fractions on cytokine production by bovine innate immune cells.

(A) IL - 10, (B) IL - 12, (C) MIP - 1β, (D) TNFα & (E) IL - 6. Points represent mean responses from duplicate wells for each of 7 animals tested. Lines indicate that cells were derived from the same animal; * 0 < 0.05; ** p < 0.01; *** p < 0.001

using Friedman repeated measures ANOVA with Dunn’s multiple comparisons test.

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Levels of MIP - 1β were also found to be significantly increased after exposure to the

polar lipid fraction with no significant increase seen after apolar lipid stimulation (figure

4:3 C). Both cultured monocytes and MDDC produced noticeably more MIP - 1β than

fresh monocytes, with MDDC from 6, and cultured monocytes from 3, of the 7 animals

responding strongly (figure 4:3 C).

Significant increases in TNFα production were also seen, again in response to the polar

lipid fraction (figure 4:3 D). While polar lipid treated cultured monocytes from all 7 cattle

produced significant levels of TNFα, considerably more TNFα was produced by MDDC

(figure 4:3 D). Further, the level of TNFα production was similar between fresh and

cultured monocytes (figure 4:3 D).

The production of IL - 6 (figure 4:3 E) followed a broadly similar pattern to that of TNFα

(figure 4:3 D) although statistical significance was not achieved. Fresh monocytes from 2

cattle produced more IL-6 after exposure to the polar lipids, which also drove increased

IL-6 production in cultured monocytes from 6 cattle (figure 4:3 E). Polar lipid driven IL - 6

production by MDDC was noted in 5 of the 7 animals screened, with one of these animals

producing more IL - 6 to the apolar lipid fraction than the polar. While statistical

significance was not achieved, the levels of IL - 6 produced by MDDC are notably higher

than from fresh or cultured monocytes (figure 4:3 E).

These data clearly demonstrate that the polar lipid fraction drives the production of

significant amounts of IL - 10, IL - 12, MIP - 1β and TNFα from all cell types. Furthermore,

it is clear that MDDC produced more IL - 12, TNFα and IL - 6 than fresh or cultured

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monocytes and MIP -1β production is greater from both cultured monocytes and MDDC

than in fresh monocytes.

Phenotypic Responses to Crude Mycobacterial Lipids

Exposure of bovine antigen presenting cells to the polar lipid fraction led to

significant increases in the production of a variety of cytokines (figure 4:3 A - E), all of

which can play important roles in directing the subsequent cell - mediated response. In

order to further assess the effect of M. bovis - derived lipids and the local cytokine milieu

on these cells, analysis of the expression of key antigen presentation related molecules

was assessed by flow cytometry (figure 4:4).

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Figure 4:4 - Effect of stimulation with the polar and apolar lipid fractions on phenotype of bovine innate immune cells. (A) MHCII, (B) CD86, (C) CD1b and (D) CD40. A single point represents the median fluorescence intensity of the specific

stain after subtraction of an isotype control (ΔMFI) for each of 7 animals tested; ** p < 0.01; *** p < 0.001 using repeated measures ANOVA with Bonferroni multiple comparisons test.

Stimulation with the polar lipid fraction resulted in a significant reduction in the cell

surface expression of MHCII on all three cell types (figure 4:4 A). Furthermore, MHCII

expression was also lower on MDDC following stimulation with apolar lipids, although this

did not achieve statistical significance (figure 4:4 A). CD86 expression was also

significantly reduced on all three cell types following stimulation with the polar lipid

fraction (figure 4:4 B). While there was a trend for lower CD86 expression on all three

cell types following stimulation with the apolar lipid fraction, this again did not achieve

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statistical significance (figure 4:4 B). Similarly to unstimulated cells, monocytes and CM

stimulated with either lipid fraction did not express CD1b (figure 4:4 C). However,

incubation of MDDC with the polar lipid fraction resulted in a significant reduction in

CD1b surface expression (figure 4:4 C). Not all cell surface molecules were down -

regulated following treatment with lipids. CD40 expression on both monocytes and

cultured monocytes increased significantly following stimulation with the polar lipid

fraction (figure 4:4 D), although no effect was seen on MDDC. Finally, no significant

difference was seen in CD80 levels following stimulation with either the polar or apolar

lipid fractions (data not shown).

In summary, these data demonstrate that M. bovis - derived lipids, and in particular the

polar fraction, downregulate the expression of several key cell surface molecules involved

in antigen presentation.

Consequence of MDDC Exposure to M. bovis - Derived Lipids

To identify and assess any functional consequence of the lipid induced reduction

in molecules related to antigen presentation the ability of lipid treated innate cell types

to stimulate an alloreactive response was assessed. Co - culture of the responder PBMC

population with either untreated MDDC or cultured monocytes resulted in a 5 - fold

increase in their proliferation (figure 4:5). No proliferation was noted for either MDDC or

cultured monocytes in the absence of responder cells (data not shown). Polar lipid

treated MDDC retained their ability to induce proliferation in the responder population

despite the downregulation of important costimulatory molecules. In contrast, allo -

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stimulation of the responder population by polar lipid treated cultured monocytes

resulted in significantly reduced proliferative responses to levels comparable with the

unstimulated responder control cells (figure 4:5).

Figure 4:5 - Proliferative responses of PBMC stimulated with polar lipid treated allotypic cultured monocytes and MDDC Red bars - polar lipid treated allotypic cultured monocytes or MDDC; light grey bars - untreated allotypic cultured

monocytes or MDDC. Bars represent the mean of triplicate wells ± standard error of the mean; *** p < 0.001 using repeated measures ANOVA with Bonferroni multiple comparisons test.

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Discussion

To assess whether crude lipid fractions were capable of mediating the responses

of bovine innate cells, stimulation experiments were performed and the level of a range

of cytokines was analysed. Cells were stimulated with either polar and apolar fractions,

however it was only after stimulation with the polar fraction that significant increases in

the production of various cytokines were detected. Perhaps most striking is the

significant increase in the production of both the Th - 1 polarising IL - 12 and the anti-

inflammatory cytokine IL - 10 (figure 4:3 A - B). Initially this seems contradictory, but it is

important to note that the fractions used are complex mixtures of a variety of lipids some

of which, such as MMG(115), are known to induce potent immunostimulatory cytokine

profiles while others, such as glycerol monomycolate (GroMM), are known Th2

polarisers(290). Further, many lipid molecules from M. tuberculosis have been shown to

mediate a range of pleiotropic effects on innate cells in murine models(291, 292).

There is little information in the published literature discussing cytokine production by

lipid treated antigen presenting cells. Instead, much work concentrates on assessing

CD4+ and CD8+ T cells responses either after direct exposure of the lymphocytes to lipid

antigens or by stimulating lymphocytes with infected or treated antigen presenting cells.

For example, TDM, which is present in the polar and apolar fractions (figure 3:1 D - E), has

been shown to induce both Th1 and Th2 cytokines. The induction of IFNγ and IL - 12 and

the depletion of IL - 4 producing NK cells has been attributed to TDM(293, 294) as well as a

role, along with IL - 6 and TNFα, in stable granuloma formation(295). Yet TDM is also

implicated in the production of IL - 5 and IL - 10 in a CD1 - dependant manner(296).

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Furthermore, GroMM has been implicated in the induction of Th2 polarising

responses(290) whilst the closely related GMM has been shown to induce Th1 cytokine

responses in T cells(297). Similarly, anti - inflammatory effects have also attributed to PIM2

and PIM6 where, upon lipid treatment of LPS activated macrophages, Doz et al. measured

downregulation of TLR4, TNFα, IL - 12p40, IL - 6, KC and IL - 10 as well as MyD88 mediated

NO release(298). However, PIM2 has also been shown to activate MDDC, inducing

significant increases in IL - 12 production(299, 300).

Given the significant increase in IL - 10 production by all innate cell types assessed, and

the important role these cells play in generating and directing the immune response, the

expression of antigen presentation associated cell surface molecules after lipid exposure

was analysed. Lipid treatment of APCs lead to a significant decrease in the levels of

costimulatory molecules associated with antigen presentation including MHCII and CD86

on all cell types studied and CD1b on MDDC (figure 4:4 A - C). Negative regulation of

these molecules by a variety of lipid components has been noted previously, especially

MHCII in human and murine systems. Similar to the data presented here, the 19 - kDa

lipoprotein is capable of downregulating MHCII expression on human THP - 1

macrophages by inhibiting activation of the IFNγ - induced CIITA(301, 302). Downregulation

of MHCII, as well as TLR2 and TLR4, has also been reported on human MDDC after lipid

exposure(303) and a further study also found impaired expression of CD1a, MHCII, CD80

and CD83 on human MDDC(304).

Downregulation of CD1 molecules has also been shown previously. Gagliardi et al.

demonstrated that MDDC generated in vitro from monocytes which had been treated

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with BCG did not express CD1 and showed reduced MHCII, CD40 and CD80(305) and this

has since been shown to be due to cell wall associated carbohydrate α - glucan(225) and

mediated through the p38 MAPK pathway(306). However these experiments have all been

performed in human or murine systems and with specific lipids, often from avirulent

bacterial isolates.

Interestingly, treatment of fresh and cultured monocytes with the polar lipid fraction

significantly increased the level of CD40 expression (figure 4:4 D) and this effect is not

seen on MDDC. This finding seems contradictory to the published literature(305, 307)

however these studies used avirulent BCG or TDM alone, rather than the complex and

more biologically representative lipid preparations derived from virulent mycobacteria

used here, as well as being performed in human or murine macrophage models. Bovine

MDDC expression of CD40 does not alter after stimulation with either polar or apolar

lipids which may be due to its constitutively higher levels of expression than on fresh or

cultured monocytes.

Finally, no difference was seen in the expression of CD80 after lipid treatment, although

this has also been reported in other systems using virulent M. tuberculosis or avirulent

BCG derived lipids(304, 305, 307).

The significant reduction in the levels of MHCII, CD86 and CD1b is consistent with the

phenotype of an impaired antigen presenting cell(308). Given the effect of the polar lipids

on the expression of these molecules and the concurrent increase in IL - 10 production, it

was hypothesised that the polar lipid fraction, or one of its components, hampers the

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ability of the cells to successfully present antigen to T cells and may be able to suppress

the induction of a Th1 response during infection. To assess any functional deficit in these

cells, especially due to the loss in MHCII, lipid treated and untreated cells were used to

drive allotypic proliferative responses.

Cultured monocytes drove proliferation of allogeneic PBMC (figure 4:5) and treatment of

cultured monocytes with the polar lipid fraction significantly abrogated these responses

as suggested by the downregulation of MHCII and other costimulatory molecules.

Proliferative responses were also seen when allogeneic PBMC were combined with

untreated MDDC (figure 4:5) however no difference in proliferation was seen using lipid

treated MDDC despite flow cytometric analysis revealing characteristic reduction in the

level of MHCII on the MDDC (data not shown). While these results seem at odds with

each other, it is possible that the loss of MHCII may be overcome by the high level of

CD40 expressed by MDDC (figure 4:4 D) or the constitutively higher levels of IL - 12

produced by these cells which further increases significantly after lipid stimulation (figure

4:3 B). Also, some evidence exists that the presence of CD80 is enough to stimulate

allogeneic T cells in the absence of CD86 signalling(309). Given the significant reduction in

CD86 expression on MDDC, the maintenance of CD80 may play a role. Finally, it is

possible that, due to constitutively higher levels of MHCII and CD40 present on MDDC, as

well as their expression of CD1b, the levels of MHCII and CD86 on these cells remains

sufficient to drive an allotypic reaction.

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These data demonstrate that M. bovis derived lipid fractions are capable of stimulating

responses in bovine innate cells and that these different cell types respond in distinct

ways.

Interestingly, the alteration in cell surface phenotype of both cultured monocytes and

MDDC seen after polar lipid stimulation is also evident after exposure to the apolar lipid

fraction, albeit to a lesser, not statistically significant, extent. This may be due to specific

lipid components present in both the polar fraction and the apolar preparation, such as

TDM. However, it may also be due to the insolubility of less polar lipids in the aqueous

environment an in vitro culture system which may limit lipid bioavailability.

Despite the presence of individual compounds known to have Th1 inducing properties,

the overall effect of these fractions appears to be down modulation of APC function. This

may be a strategy employed by the bacilli to delay protective immune responses. The

effects presented here confirm similar to observations seen in humans(302, 310-315). In

conclusion, this chapter demonstrates the ability of the lipid fractions to mediate effects

on bovine innate cells. These lipids, especially those contained within the polar fraction

are capable of interacting with the host’s innate immune cells such that the cells ability to

initiate an adequate T cell response may be compromised, although this effect could only

be demonstrated for cultured monocytes and not MDDC. The effects mediated by these

lipids may play a pivotal role in the outcome of infection and subfractionation may help

to differentiate down - modulatory compounds from those that may have adjuvant or

Th1 stimulatory properties.

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Chapter Summary

• Objective

To measure any effects mediated by the lipid fractions on bovine innate

immune cells, characterise the innate cell cytokine and phenotypic responses

and assess if the function of the cell was affected.

• Results

MDDC and CM were derived and characterised. CM were found to express

significantly increased levels of CD80 and no CD1b; MDDC were found to

express high levels of CD1b and CD40.

The polar fraction was found to drive significant increases in the levels of IL -

10, IL - 12, MIP - 1β and TNFα from all cell types. MDDC were seen to produce

more IL - 10 than other cells. Polar lipids were also shown to significantly

decrease expression of MHCII and CD86 on all cell types as well as CD1b on

MDDC. Polar lipids also significantly increased CD40 expression by monocytes

and CM.

Polar lipid treated CM drove significantly less proliferation in allogeneic PBMC

than untreated CM. This effect was not seen with MDDC.

• Conclusions

The polar lipid fraction has been shown to modulate both the cytokine

production and potential antigen presentation ability of bovine innate immune

cells. These lipids may play a critical role in defining the outcome of infection.

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Chapter Five Effect of Lipid Subfractions on Bovine Innate Immune Cells

Chapter Five

Effects of Lipid Subfractions on Bovine Innate Immune Cells

Background

It is clear that the components in the polar lipid fraction are capable of interacting

with bovine innate immune cells in a manner which may impair the cells effectiveness in

modulating immune responses. These effects could mean that the innate cells ability to

initiate an adequate T cell response may be compromised. However, the use of crude

lipid fractions containing the total free extractable lipid from M. bovis does not allow for

any discrimination of effects mediated within the antigen mixtures and the responses

seen so far cannot be attributed to any specific lipid entities.

Individual lipids have been shown to bind with a large array of innate cell receptors and

mediate a broad range of effects. Various mycobacterial lipids have been shown to be

recognised directly by TLR2 including AraLAM(175), lipomannan(176), PIM2(175)

, and PIM6(177)

or in association with TLR1 / 6, TLR4(316, 317) or other molecules.

For example, Means et al. demonstrated the TLR2 mediated recognition of LAM but

found that CD14 was required for signalling and effector function modulation(318) whilst

Jones et al. showed that TLR2 mediated recognition of PIM2 generated increases in TNFα

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as well as activation of the NF - κB and MAP kinases without the requirement for

accessory molecules(175). In another study, Means et al. also demonstrated CD14

independent, TLR4 - mediated activation of both CHO cells and murine macrophages by a

heat labile, protease resistant cell wall component able to induce TNFα production(319).

Despite further research demonstrating that TNFα production can be blocked by a TLR4

antangonist, this cell wall component remains unidentified(320) and most TLR - activating

purified mycobacterial antigens tested so far signal through TLR2(317).

Polar lipids such as PIMs are also known to mediate a range of effects based on

differential receptor binding. Although both PIMs and LM contain mannose and have

been shown to regulate cytokine, NO and T cell responses(321-323), LM has been shown not

to bind to the mannose receptor but to DC - SIGN and mediate a responses through

TLR2(324). Through this mechanism, LM induces apoptosis(325) and IL - 12 production by

macrophages(326). In contrast to LM, the higher PIMs (PIM5-6) have been shown to bind to

the mannose receptor and can interfere with phagosome : lysosome fusion(183) whilst the

lower mannosylated PIMs (PIM1-4) preferentially bind to complement receptor 3 (CR3)

and facilitate phagosome fusion with the early endosomal compartment(327-329). To

further complicate the situation, more recent work has shown that the higher

mannosylated PIMs are also bound by DC - SIGN(330).

Unsurprisingly, lipid recognition and receptor interaction exhibits a degree of cellular

specificity. For example, human MDDC have been repeatedly shown to bind M.

tuberculosis through DC - SIGN(182, 331, 332) whilst the major M. tuberculosis receptors on

macrophages are CR3 and the mannose receptor(183, 327-329).

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The huge complexity highlighted here demonstrates both the variety of potential

antigenic structures on the bacterial surface as well as the broad array of host molecules

which play a role in host - pathogen interaction. The crude lipid extracts used previously

are enormously complex and contain an array of lipid molecules shown here to mediate a

broad range of effects on the host, as well as some lipopeptide and unidentified lipids.

This chapter addresses the hypothesis that the polar lipid fraction could be separated into

smaller and more defined subfractions which could be used to stimulate bovine MDDC to

identify an individual or subset of lipids responsible for driving the responses seen in

Chapter Four. A variety of strategies were employed to separate this fraction into smaller

fractions or individual components. These subfractions were then used to stimulate

bovine innate immune cells and subsequently assessing the cells for changes in cytokine

production and surface phenotype.

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Results

The Effect of Crude Polar Lipids from M. bovis AN5

Due to the biosafety constraints of culturing large volumes of M. bovis AF 2122/97

and the substantial length of time required to grow the large amount of cell mass

required for subfractionation, an alternative source of material was used for these

studies. Given that M. bovis AN5 lipids appear qualitatively very similar to those from AF

2122/97 (see Chapter Three), a pellicle of AN5 was obtained. Used in the production of

bovine PPD, M. bovis AN5 cultured in this way yields very high bacterial mass. However,

as M. bovis AN5 is genotypically distinct from AF 2122/97, and it is known that conditions

of bacterial culture can affect the overall lipid profile of the cells(278, 333), the effects

mediated by the polar lipid fraction isolated from M. bovis AF2122/97 on bovine MDDC

was compared to the polar lipid fraction from M. bovis AN5.

The polar lipids from AN5 were assessed to see if they generated similar phenotypic

changes to those seen with AF 2122/97 - derived lipids (figure 5:1). As was seen

previously, treatment of bovine MDDC with AF 2122/97 - derived lipids lead to a

significant reduction in the expression of both MHCII (figure 5:1 A) and CD1b (figure 5:1

B). Despite the quantitative differences in the lipid profiles of both strains, polar lipids

extracted from M. bovis AN5 also generated a significant reduction in the expression of

MHCII (figure 5:1 A) and CD1b (figure 5:1 B). These data therefore suggested that the

AN5 - derived lipid preparation contained the same immunologically active lipid species

101


as the AF 2122/97 - derived lipid fraction and could, therefore, serve as starting material

for large scale subfractionation.

Figure 5:1 - Effect of polar lipids from M. bovis AF 2122/97 and AN5 on phenotype of bovine MDDC. (A) MHCII, (B) CD1b. Points represent mean responses from duplicate wells for each of 14 animals tested. Lines

indicate the sample median; * p < 0.05; *** p < 0.001 using Friedman repeated measures ANOVA with Dunn’s multiple comparisons test.

Subfractionation of the Crude Polar Lipids from M. bovis AN5

Having shown that the polar lipids derived from M. bovis AN5 mediate

comparable responses in bovine MDDC as those from AF 2122/97, AN5 - derived lipids

were used for subfractionation experiments. Several subfractionation methods were

trialled. Firstly, silica columns packed in CHCl3 with a mobile phase increasing in polarity

by the addition of increasing levels of CH3OH yielded no separation of the polar fraction.

As can be seen in figure 5:2 A, most lipid was eluted from the column with no polar

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solvent added (0 % CH3OH) whilst the remaining lipid eluted as soon as any CH3OH was

present (figure 5:2 B). Further, the efficiency of this method was poor. Columns were

initially loaded with 50 mg of crude lipid fraction and less than 800 µg of total lipid was

eluted, leading to an efficiency of only 1.6 %.

Figure 5:2 - Subfractionation by glass column chromatography. Crude polar lipid was loaded onto a glass column packed with silica in CHCl3 before being eluted with a mobile phase

consisting increasing levels of CH3OH. TLCs run in solvent system E and stained with MPA. (A) 0 % v/v; (B) 2 % v/v; (C) 5 % v/v; (D) 10 % v/v; (E) 15 % v/v; (F) 20 % v/v; (G) 30 % v/v; (H) 50 % v/v.

This method was refined with the use of syringe mounted solid phase extraction columns.

Columns were loaded with 5 mg of AN5 - derived crude polar lipid and the same mobile

phases applied. Some lipid material was washed from the column even in the presence

of no polar solvent (figure 5:3 A). Once CH3OH was present, increasing the polarity of the

mobile phase had little effect until the level of solvent reached 5 % where

phosphatidylinositol and diphosphatidyl glycerol began to elute off the column (figure

5:3 C). However, once the polarity of the mobile phase was increased to 15 % CH3OH

(figure 5:3 E) all the components of the crude fraction were eluted from the column.

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Increasing the levels of CH3OH beyond this point increased the elution of PIMs and the

more polar components of the fraction (figure 5:3 F - H).

Figure 5:3 - Subfractionation by solid phase extraction chromatography. Crude polar lipid was loaded onto a solid phase extraction column packed with silica in CHCl3 before being eluted with a

mobile phase consisting increasing levels of CH3OH. TLCs run in solvent system E and stained with MPA. (A) 0 % v/v; (B) 2 % v/v; (C) 5 % v/v; (D) 10 % v/v; (E) 15 % v/v; (F) 20 % v/v; (G) 30 % v/v; (H) 50 % v/v.

Whilst this method allowed for some discrimination of the lipids within separate

fractions, the carryover of lipids between subfractions was quite marked (figure 5:3). The

use of SPE columns was found to be more efficient that silica column chromatography.

Of the 5 mg used for each experiment, around 0.5 mg of lipid was eluted in the combined

subfraction. This equates to an efficiency of around 10 %.

To try to further separate the polar fraction, and decrease loss of the crude lipid fraction,

the lipids were separated by 1 dimensional TLC, visualised under UV light after staining

with 1, 6 - diphenyl - 1, 3, 5 - hexatriene (figure 2:2). Individual bands were scraped from

the plate and the lipid dissolved into a 2 : 1 mixture of CHCl3 : CH3OH. Using this

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approach, it was possible to isolate 6 individual subfractions as can be seen in figure 5:4.

Each scraped band corresponds to a region of the polar lipid fraction and little carryover

between subfractions is visible (figure 5:4).

Figure 5:4 - One dimensional TLC of the polar lipid subfractions. Crude polar lipids were separated by TLC and 6 bands scraped off TLC plates and pooled into individual subfractions.

Each individual band can be seen here run in the same 1D TLC system next to the polar fraction for comparison.

Further analysis of these subfractions by 2D TLC was performed using 2D TLC system E

(figure 5:5). Band 1 was found to contain phospholipid, phosphatidylinositol, Ac1PIM2

and Ac2PIM2 (figure 5:5 A). P, PI and Ac2PIM2 were also found in band 2, as well as DPG

(figure 5:5 B). Band 3 contained Ac2PIM2, DPG and PE (figure 5:5 C) whilst bands 4 and 5

both Ac2PIM2 and PE and DPG respectively (figure 5:5 D and E). Band 6 was initially

scraped from the solvent front of the 1D TLCs and contained no identifiable lipids (figure

5:5 F).

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Figure 5:5 - 2D TLC analysis of the 6 polar lipid subfractions stained with MPA (A) Band 1, (B) Band 2, (C) Band 3, (D) Band 4, (E) Band 5, (F) Band 6. Each subfraction was run on 2D TLC system E.

The subfractions generated by 1D TLC were demonstrably different in their lipid

compositions and exhibited less carryover between subfractions than using either of the

column based methods. Furthermore, the efficiency of subfractionation was increased

greatly. Of the 30 mg loaded onto each 1D plate, the combined subfractions contained at

least 6.5 mg which equates to an efficiency of approximately 21 %. Given the greater

separation between the subfractions and increase in efficiency, 1D TLC subfractionation

was chosen as the method to generate subfractions for further biological analysis.

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Cytokine & Phenotypic Responses to Lipid Subfractions

Having successfully separated the polar lipid fraction, the 6 discrete subfractions

were tested for their ability to modulate MDDC functions as seen with unfractionated

polar lipids (see Chapter Four). MDDC were generated from 3 uninfected cattle and

stimulated with the AN5 - derived polar lipid fraction and each of the 6 subfractions at 20

µg ml-1 and the levels of IL - 10 and IL - 12 were measured as described above.

The AN5 - derived polar lipid fraction generated robust IL - 10 responses from the MDDC

of all 3 animals tested with a median IL - 10 level of 66,404 RLU compared to a median Nil

of 26,553 RLU (figure 5:6 A). All 6 subfractions also drove increases in IL - 10 production

with bands 3 and 4 generating the highest responses while bands 1 and 6 generating the

lowest responses although this trend was not significant (figure 5:6 A). Similarly, an

increase in IL - 12 production was seen after lipid stimulation of the MDDC (figure 5:6 B).

The AN5 - derived polar lipid fraction increased the median IL - 12 response and

responses of a similar magnitude were seen after stimulation with bands 2, 3 and 4 while

bands 1, 5 and 6 drove more modest increases in IL - 12. Again, this trend was not

statistically significant.

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Figure 5:6 - Effect of polar lipids from M. bovis AN5 on cytokine production by bovine MDDC (A) IL - 10, (B) IL - 12. Points represent mean responses from duplicate wells for each of 3 animals tested.

Measurements in Relative Light Units (RLU). Lines indicate the sample median; ns = not significant using Friedman repeated measures ANOVA with Dunn’s multiple comparisons test.

MDDC expression of MHCII and CD1b after stimulation with the lipid subfractions was

also assessed (figure 5:7). Stimulation of MDDC with AN5 - derived polar lipid fraction

generated a notable loss of MHCII expression with the median Δ MFI decreasing from

243.08 to 55.55 (figure 5:7 A). Again, all the subfractions mediated a similar effect in

reducing the level of MHCII expression to almost the same level with the median Δ MFI

ranging from 59.71 for band 5 to 68.15 for band 6 (figure 5:7 A).

MDDC expression of CD1b after stimulation with the lipid subfractions followed a similar

pattern to that seen for MHCII. Exposure of the cells to the polar lipids generated a

noticeable loss of surface CD1b (figure 5:7 B) where the median Δ MFI of 16.03 for

untreated cells dropped to 3.13. Treatment of MDDC with any of the subfractions also

caused a loss of CD1b expression with the median Δ MFI ranging from 4.89 for band 4

down to 2.23 for band 5 (figure 5:7 B).

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Figure 5:7 - Effect of lipid subfractions from M. bovis AN5 on phenotype of bovine MDDC (A) MHCII, (B) CD1b. Points represent mean responses from duplicate wells for each of 3 animals tested. Lines indicate

the sample median; * p < 0.05; ns = not significant using Friedman repeated measures ANOVA with Dunn’s multiple comparisons test.

As each subfraction contained fewer lipid moieties than the crude fraction, it was

possible that their use at the same concentration as the crude fraction (20 µg ml-1) meant

that individual lipids were present at higher concentrations in the subfractions than in the

crude fraction, and this could have been one possible reason for the lack of

discrimination between the subfractions. To assess this, MDDC from 2 animals were

exposed to the subfractions at a series of concentrations. Again, exposure of bovine

MDDC to the polar lipid fraction from AN5 generated elevated levels of IL - 10 (figure 5:8).

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Figure 5:8 - Effect of serially diluted polar lipids from M. bovis AN5 on IL - 10 production by bovine MDDC Points represent mean responses from duplicate wells for each of 2 animals tested. Solid lines indicate the sample

mean; dashed lines indicate the Nil or AN5 Polar mean.

Stimulation of bovine MDDC using bands 1 - 5 also drove dose dependant increase in IL -

10 production (figure 5:8). Band 6 did not increase in the level of IL - 10 produced

regardless of the concentration at which it was used (figure 5:8). Treatment of cells with

band 1 - 5 at 20 µg ml-1 drove IL - 10 production but, in general, not to the same level as

the AN5 - derived polar lipid fraction alone (figure 5:8). Of the subfractions, band 4 drove

the largest IL - 10 response (figure 5:8). Overall no increases were seen when any of the

subfractions were used at 1 µg ml-1 (figure 5:8).

An increase in production of IL - 12 was also evident after stimulation with the crude AN5

- derived polar fraction and bands 1, 2 and 4 (figure 5:9). Again, the increase in IL - 12

production in response to the lipid subfractions was dose dependant and the level of IL -

12 detected when bands 1, 2 and 4 were used at 1 µg ml-1 had returned to the

background level (figure 5:9). Unlike IL - 10 production, those subfractions which

stimulated IL - 12 production did so to at least a similar level as the complete AN5 -

derived polar lipid fraction (figure 5:9). In fact, the mean level of IL - 12 driven by band 4

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was higher than that for the AN5 polar fraction (figure 5:9). Bands 3, 5 and 6 induced

limited increases in IL - 12 from bovine MDDC (figure 5:9).

Figure 5:9 - Effect of serially diluted polar lipids from M. bovis AN5 on IL - 12 production by bovine MDDC Points represent mean responses from duplicate wells for each of 2 animals tested. Solid lines indicate the sample


As the decrease in MHCII expression had been a consistent indicator of lipid activity on

cell phenotype, this was also assessed with the diluted lipid subfractions. As expected, a

large reduction in MHCII expression was seen after stimulation with the crude polar

fraction from AN5 (figure 5:10). Further, all of the lipid subfractions also caused a

decrease in the levels of MHCII expressed, although this reduction was not of the same

magnitude as the crude lipid fraction (figure 5:10). The reduction of MHCII levels was

also dose dependant, with the expression of MHCII increasing as the concentration of

lipid subfraction decreased. When the subfractions were used at 1 µg ml-1 the levels of

MHCII were similar to that of unstimulated cells (figure 5:10).

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Figure 5:10 - Effect of serially diluted polar lipids from M. bovis AN5 on MHCII expression by bovine MDDC Points represent mean responses from duplicate wells for each of 2 animals tested. Solid lines indicate the sample


Despite the differences in lipid composition of the subfractions, when used to stimulate

MDDC, little difference was seen in their ability to drive cytokine responses or affect

levels of MHCII expression. Band 4 drove the highest mean levels of IL - 10 and IL - 12

production and had the strongest effect on mean MHCII expression, however the

responses generated by each subfraction were broadly similar. As the biological activity

of the subfractions was dose dependant the subfractions were assessed for the presence

of other potentially stimulatory molecules.

Assessment of Lipopeptide presence in Lipid Subfractions

Subfractionation of the M. bovis AN5 - derived polar fraction generated 6

subfractions which contained demonstrably different lipid compositions (figure 5:5).

When bovine MDDC were treated with these subfractions, bands 1 - 5 were able to

generate dose - dependent increases in the levels of IL - 10 (figure 5:8) and all bands

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generated increased levels of IL - 12 (figure 5:9). Further, all bands led to a dose

dependant reduction in the expression of MHCII (figure 5:10). However there was little

difference between the responses to the different subfractions despite the differing lipid

contents of the subfractions.

To try to explain the lack of variation in biological responses to these subfractions, each

was re - analysed by 2D TLC and stained with ninhydrin to assess the presence and

distribution of any lipopeptide.

Figure 5:11 - 2D TLC analysis of the 6 polar lipid subfractions stained with ninhydrin (A) Band 1, (B) Band 2, (C) Band 3, (D) Band 4, (E) Band 5, (F) Band 6. Each subfraction was run on 2D TLC system E. Red

arrows indicate primary amines; yellow arrows indicate secondary amines; black arrows indicate charred lipid for reference. Identifiable spots are named.

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Amine residues were found throughout the subfractions. As was seen in the crude lipid

fractions (figure 3:2 and figure 3:4) a primary amine containing molecule was identified

co - located with PI and was visible in bands 1, 2 and 4 (figure 5:11 A, B and D). Band 1

also contained 4 unidentified secondary amines (figure 5:11 A). As well as the primary

amine located at the PI spot, another primary amine of unknown identity and several

unknown secondary amines were found (figure 5:11 B). In band 3, a primary amine was

identified located very close to the DPG spot along with 2 more unknown molecules

(figure 5:11 C). Band 4 contained the primary amine located at the PI spot and 4

unidentified secondary amine residues (figure 5:11 D). Band 5 also contained amine

residues, 1 primary and 1 secondary, neither of which were co - located with lipid

molecules (figure 5:11 E). Finally, band 6 contained no clear amine resides, however a

secondary amine residue was identified as a smudge (figure 5:11 F).

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Discussion

In an attempt to identify individual lipid components responsible for the effects

mediated by the polar fraction in bovine innate immune cells, the polar fraction from M.

bovis AN5 was subfractionated and the subfractions analysed and tested. The use of AN5

for subfractionation was primarily due to biosafety concerns over the cultivation of large

volumes of AF 2122/97 when a pellicle of AN5 was already available. Further, analysis of

the lipids from M. bovis AN5 showed little difference when assessed by MPA (figure 3:1

and figure 3:3) and ninhydrin stained TLCs (figure 3:2 and figure 3:4).

Despite the similarities when analysed by TLC, it was important to assess the ability of the

AN5 - derived fraction to mediate similar responses seen to the AF 2122/97 - derived

lipids. To this end, both polar lipid fractions were used to stimulate bovine MDDC and

the effect on expression of MHCII and CD1b was assessed.

As was seen previously (figure 4:4), stimulation of bovine MDDC with both the AF

2122/97 and AN5 - derived polar lipids generated a significant reduction in the expression

of MHCII (figure 5:1 A). Only the AF 2122/97 - derived polar fraction was capable of

driving a significant reduction in the levels of CD1b (figure 5:1 B), however a clear trend

for reduced CD1b is evident after stimulation with the AN5 - derived polar fraction.

Overall both the effector and phenotypic responses to the AN5 - derived polar lipid

fraction were similar to that seen with the AF 2122/97 - derived lipids and the AN5 -

derived fraction was used for subfractionation.

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Initially subfractionation was attempted using column chromatography. Silica packed

columns were run in CHCl3 with increasingly polar eluents, made by the addition of

increasing proportions of CH3OH. This method of fractionation, and variations there on,

have been used in a variety of settings and with much success(334-336), however this

method failed to separate the AN5 - derived polar lipid fraction. Despite the lack of any

polar solvent in the first eluent, the entire fraction was visible in this elute (figure 5:2 A).

The addition of 2 % CH3OH caused the elution of the rest of the fraction (figure 5:2 B) and

no further lipid material appeared in any subsequent elutions (figure 5:2 C - H). The lack

of separation could be related to the flow rate of the column. If this was too fast, the

column would not have fully equilibrated and the lipid fraction would be forced through

the column. This could also explain the fact that of the 50 mg of polar fraction loaded

onto the column, less than 1 mg of material was eluted from the column.

Although large scale column chromatography failed to generate any discriminate

subfractions and was found to be extremely inefficient, many authors have reported

successful column chromatographic separation of polar lipids(334-336). As a refinement,

silica packed SPE columns were used with smaller starting quantities of AN5 - derived

polar fraction. While this method did allow for the elution of PI and DPG in 5 % CH3OH

(figure 5:3 C) and the PIMs, PI and PE at 50 % (figure 5:3 H) there was no discrimination

between the subfractions. Again, other authors have reported successful use of SPE

columns to separate lipids, including those from M. bovis BCG(337) and M. smegmatis(338),

however separation of M. bovis BCG lipids required magnesium silicate columns, more

complex solvent systems and further purification by anion - exchange

chromatography(338). The separation of M. smegmatis polar lipids using SPE columns also

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used a magnesium silicate stationary phase followed by separation by 2D TLC and reverse

- phase chromatography(337).

Rather than pursue more complex column separation techniques, the separation of lipids

by TLC and their subsequent removal from the plate was ultimately used for separation.

A variety of TLC systems have been employed for purification, many with great

success(339-342). One dimensional TLC allowed the identification of 6 bands which could be

individually collected, purified and analysed (figure 5:4). Subsequent 2D analysis of each

subfraction demonstrated discrete fractions with minimal carryover of lipid molecules

between subfractions (figure 5:5).

Having generated 6 individual polar lipid subfractions, these were tested in the same

innate cell systems described previously. Measurement of IL - 10 and IL - 12 showed

increased levels of both cytokines (figure 5:6). Assessment of the levels of MHCII and

CD1b after stimulation with the subfractions also showed reductions in the expression of

both molecules but no differences between the subfractions were observed (figure 5:7).

The use of the subfractions at a single concentration could mask responses as some lipid

species were present in more than one subfraction, and at different concentrations. To

address this, the subfractions were serially diluted and tested again. Interestingly,

measurement of IL - 10 production by bovine MDDC showed that after stimulation with

the 6 serially diluted subfractions, all except band 6 (the solvent front from the 1D TLC

separations) drove dose dependant increases which returned to the background level as

the fractions were diluted to 1 µg ml-1 (figure 5:8). None of the subfractions drove IL - 10

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production as high as the AN5 polar fraction, suggesting that the response driven by the

complete fraction may be at least partly synergistic one, requiring components in

different subfractions (figure 5:8). No differences in the levels of IL - 10 produced was

seen between the subfractions suggesting something present in all fractions may be

responsible for driving the production of IL - 10.

In contrast, differential IL - 12 production was seen after stimulation of bovine MDDC

with the serially diluted subfractions. As seen previously, the AN5 - derived polar fraction

drove increased IL - 12 production as did bands 1, 2 and 4. Further, the responses seen

after exposure to these bands were dose dependant and IL - 12 levels returned to the

background when the subfractions were used at 1 µg ml-1 (figure 5:9). The highest levels

of IL - 12 were seen in response to subfractions 1 and 4. Band 1 consisted of mostly the

lower mannosylated PIMs and other phospholipids (figure 5:5 A) which are known to

drive IL - 12 production from DC in vitro(330). Interestingly, the only identifiable lipid in

band 4 was PE (figure 5:5 D) and no inflammatory effects have been attributed to this

molecule.

Although 2 subfractions generated slightly different IL - 12 responses, the lack of

discrimination between the subfractions based on their ability to drive cytokine

production was disappointing. To try to further differentiate the subfractions, the levels

of MHCII expression were assessed after exposure of bovine MDDC to the serially diluted

subfractions. Again, each subfraction drove a reduction in the levels of MHCII present on

the cells and expression was regained as the subfraction was diluted, but no differences

in the magnitude of the effect were seen between the subfractions (figure 5:10).

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Overall, no differences between the subfractions were seen in their ability to mediate the

responses characterised originally using the crude polar fraction, despite the fact that

each fraction clearly consisted of different lipid molecules (figure 5:5). These data

suggested some other component, or components, present in the subfractions which had

not been identified. As lipopeptide had been identified in the crude polar fractions from

both AF 2122/97 (figure 3:2) and AN5 (figure 3:4) , ninhydrin stained 2D TLCs were

performed to assess the presence of any remaining lipopeptide in the subfractions (figure

5:11).

As can be seen in figure 5:11, lipopeptide could be identified in all 6 subfractions, albeit to

a lesser extent in band 6 (figure 5:11 F). Interestingly ninhydrin staining residues could be

seen which were not apparent when the crude fractions had been analysed (indicated

with red arrows in figure 5:11 A - F). It is most likely that these molecules were present in

the crude fraction but not visible due to their lower concentration. The degree of

lipopeptide overlap between the subfractions suggests that the lipopeptide molecules

migrated across the entire 1D TLC plate during the initial separation. Lipopeptides are

known to mediate a range of effects on the immune system which may account for the

apparent lack of lipid specificity seen here. Despite knowledge of mycobacterial

lipopeptides, no classification or categorisation has ever been performed, hence there is

no reference work from which to identify lipopeptides. The best characterised

mycobacterial lipopeptide is the 19 kDa lipoprotein, which has been shown to mediate a

broad range of effects on innate immune cells. It is known to inhibit antigen processing

in murine macrophages by decreasing the synthesis and expression of MHCII(301, 313, 343)

and MHCI(344) as well as increasing IL - 12 production by human macrophages(345). These

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are similar to the effects seen when using the crude lipid fractions to stimulate bovine

MDDC in Chapter Four. Conversely the same lipoprotein, when cloned into M.

smegmatis, has been associated with immune suppression by a reduction in the levels of

IL - 12, IL - 10 and TNFα(346) and lipopeptides from other bacterial genera have been

shown to stimulate maturation of DC as measured by increasing expression of MHCII,

CD80 and CD86(347).

The lipopeptides present in these lipid subfractions have not been identified but

considering the degree of overlap between fractions, and the consistency of the

biological effects seen, it is not unreasonable to suggest that they may play a significant

role in the responses seen to the subfractions.

These data demonstrate that selective purification of individual lipid fractions from a

crude mixture is challenging. While the lipid content of each subfraction is demonstrably

different from the others, the biological effects they mediate on bovine MDDC do not

reflect this and no response can be attributed to any particular lipid. Lipopeptides,

surprisingly not removed by modified Bligh and Dyer extraction used to remove the silica

contamination from the subfractions, can be identified in each fraction. Further, the high

degree of lipopeptide carryover between subfractions could explain the similar biological

responses seen to the subfractions. In addition, it appears that more lipopeptide is

present in band 1 and band 4, hinting at a possible cause for the slightly stronger

responses seen to band 4, whilst less lipopeptide was present in band 6 where responses

to which were lower in magnitude or non - existent. That Ac2PIM2 was present in all

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subfractions apart from band 6 further highlights the difficulty in purifying individual lipid

molecules and this may also be important in the lack of differential responses.

Also of note is the absence of the higher mannosylated PIMs from these subfractions.

Two possible explanations the their absence exist: firstly it is possible that the 1D solvent

system did not allow for their movement from the origin, although this is unlikely as

almost no lipid remains visible at the origin; or secondly that the high degree of

mannosylation caused the molecules to locate at the interface of the modified Bligh and

Dyer extraction during the removal of colloidal silica and the PIMs could have been

removed with the aqueous phase.

In conclusion, this chapter demonstrates the complexities and challenges associated with

selective purification of individual lipid species from complex mixtures. Despite the

generation of 6 subfractions of differing lipid compositions, probably due to the extensive

overlap of lipid species across the fractions thus preventing the purification of individual

lipid species, no differences were seen in the biological responses to the subfractions.

Further analysis of the subfractions suggested that the presence of Ac2PIM2, lipopeptides

or both, could explain the consistency of the biological responses.

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Chapter Summary

• Objective

To separate the polar lipid fraction into smaller discrete subfractions which

could be characterised and used to identify which lipid or subset of lipids were

driving the responses seen previously.

• Results

The polar fractions from AF 2122/97 and AN5 were shown to mediate similar

effects on MHCII and CD1b expression by MDDC and AN5 lipids were used for

subfractionation. A variety of methods were trialled but suffered from a lack

of discrimination and poor efficiency. Finally, 1D TLC was used to generate 6

subfractions which were used for testing.

All subfractions mediated similar effects with no significant differences seen in

their ability to drive IL - 10 and IL - 12 production or the reduction of MHCII or

CD1b on MDDC. This effect was found to be dose dependent.

Further analysis of the subfractions highlighted the presence of lipopeptide in

all subfractions.

• Conclusions

The selective purification of lipid molecules from complex mixtures is

challenging. The presence of lipopeptide in the subfractions, the carryover of

Ac2PIM2 and the absence of the high mannosylated PIMs confounds the

biological responses meaning no particular response can be assigned to any

specific lipid molecule or family.

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Chapter Six Effect of Lipids on Bovine Acquired Cell - Mediated Immunity

Chapter Six

Effect of Lipids on Bovine Acquired Cell - Mediated Immunity

Background

Despite the demonstrably different lipid compositions of the lipid subfractions

and the dose dependant effects they mediated on bovine MDDC in both cytokine

production and phenotype, no differential effects were seen between the fractions.

However, a variety of amine containing molecules were identified in all of the

subfractions which suggested the presence of lipopeptide. As both lipid and lipopeptide

molecules have been shown to modulate cell - mediated immunity, the crude fractions

were assessed for the ability to generate responses in PBMC.

The identification of CD1 proteins as non - polymorphic MHCI - like molecules, and their

role in the presentation of lipid antigens to T cells(191) has led to much greater

understanding of the role of lipids in adaptive immunity. Early work demonstrated the

role of the Group 1 molecule CD1b in the presentation of mycolic acids(191) however many

other lipids have since been shown to be presented through CD1b including

sulphoglycolipids(199, 209) and glycolipids like GMM(202). CD1b is the most well understood

member of the Group 1 CD1 family thanks to work in humans but little is known about

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the other Group 1 molecules due to their genomic deletion in common laboratory

mice(348). However there has been recent interest in CD1c since the discovery that

phosphomycoketide(216) and lipopeptides containing an N - terminal acylation(215) are

CD1c restricted.

CD1d is the only Group 2 CD1 molecule, and is another well - characterised CD1 protein.

Ever since the realisation that NKT cells could respond to lipid antigens(191, 205, 349), and the

subsequent discovery of the CD1d restricted lipid antigen α - Galactosylceramide

(αGalCer)(226, 350), much research effort has been concentrated on understanding lipid

antigens. Although work was initially focussed on invariant NKT cells it has since been

shown that great diversity exists in the lipid - responsive TCR repertoire(351-353) and that

these diverse NKT cells contribute to the Th1 / Th2 balance(351, 354). It has been shown

that CD1d restricted NKT cells are capable of recognising a variety of lipid antigens

including phospholipids(227).

Recently, a CD1b restricted subset of T cells has been found(355). These cells require the

CD1B gene for their development and have been shown produce proinflammatory

cytokines in response CD1b expressing DCs(355). It is clear that NKT cells have a significant

role to play in lipid mediate responses and the hunt for their antigens has continued(224,

356, 357).

One of the most important groups of phospholipids are the PIMs. Interest in PIMs was

stimulated since it was shown that phosphatidylinositol dimannoside (PIM2) forms the

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phosphoglycolipid anchor which tethers a large array of glycolipids and lipoglycans,

including lipomannan and lipoarabinomannan, to the cellular membrane(358).

Previous analysis of the lipid fractions by ninhydrin stained 2D TLC showed the presence

of lipopeptide in the polar fractions (figure 3:2 and figure 3:4). These molecules are

known to modulate adaptive immune responses(275, 277) and have been shown to be

important targets of the MHC - restricted T cell response to M. tuberculosis(257). Further,

many lipids are known to modulate adaptive immunity(202, 203, 216, 355, 359, 360), particularly

the PIMs(208, 228, 361-363). Therefore, the polar and apolar fractions were used to assess the

role of both lipid and lipopeptide in the generation of adaptive immune responses.

This chapter addresses the hypothesis that the crude lipid fractions could be used to

generated cell - mediated immune responses and thus have potential as subunit vaccine

candidates. Further, it was hypothesised that these responses could be characterised

phenotypically and the potential role of lipid, lipopeptide or a combination of both, could

be assessed.

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Results

Lymphocyte Responses to Crude Mycobacterial Lipids

Given that both lipid and lipopeptide are known to affect cell - mediated

immunity, the crude lipid fractions were screened for their ability to drive adaptive

immune responses. The AF 2122/97 - derived polar lipid fraction drove significantly

increased proliferation (figure 6:1 A) and IFNγ production (figure 6:1 B) when compared

to the nil control. Furthermore, polar lipid stimulation drove significantly more

proliferation and IFNγ production than stimulation with the apolar lipid fraction (figure

6:1). The apolar fraction generated no increase in either proliferation or IFNγ production

(figure 6:1).

Figure 6:1 - Effect of stimulation with the AF 2122/97 polar and apolar lipid fractions on bovine PBMC. (A) Proliferation measured by 3H - Thy incorporation; (B) IFNγ production measured by Bovigam. Each point represents

the mean of triplicate wells; horizontal lines represent the sample median; * p < 0.05, *** p < 0.001 using Friedman repeated measures ANOVA with Dunn’s multiple comparisons test. PWM - Pokeweed Mitogen. Lipids used at 20 µg

ml-1 for (A) 5 days or (B) 12 - 16 hours.

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The polar lipid fraction from M. bovis AF 2122/97 was also compared to the polar fraction

from M. bovis AN5, as had been done previously for phenotype (figure 5:1) on bovine

MDDC. As seen previously, the AF 2122/97 - derived polar fraction drove a significant

increase in proliferation (figure 6:2 A), whilst the AN5 - derived polar fraction drove an

even greater increase in proliferation (figure 6:2 A). A significant increase was also seen

in IFNγ production after PBMC were exposed to the AF 2122/97 - derived polar lipids or

the AN5 - derived polar lipids (figure 6:2 B).

Figure 6:2 - Effect of stimulation with the AF 2122/97 polar and AN5 polar lipid fractions on bovine PBMC. (A) Proliferation measured by 3H - Thy incorporation; (B) IFNγ production measured by Bovigam. Each point represents

the mean of triplicate wells; horizontal lines represent the sample median; ** p < 0.01, *** p < 0.001 using Friedman repeated measures ANOVA with Dunn’s multiple comparisons test. PWM - Pokeweed Mitogen. Lipids used at 20 µg

ml-1 for (A) 5 days or (B) 12 - 16 hours.

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Lipopeptide Activity in the Crude Polar Fraction

Having shown that the polar lipid fraction from M. bovis AN5 was capable of

driving both IFNγ production and cellular proliferation in PBMC from TB infected cattle,

the effect of lipopeptide in the lipid fraction was assessed by using antibodies to block

either MHCII or CD1.

As expected, strong proliferative responses were seen to the AN5 polar fraction (figure

6:3 A, hatched bar). The addition of an isotype control antibody generated a slight

reduction in proliferation irrespective of the level of dilution of the antibody (figure 6:3 A,

grey bars). Interestingly, the addition of an anti - CD1 antibody did not affect

proliferation and responses were similar to that seen when the isotype control antibody

was added (figure 6:3 A, red bars). Further, the addition of an antibody which blocked

MHCII molecules generated a dose dependant reduction in proliferation (figure 6:3 A,

blue bars). The addition of antibody did not increase the background level of stimulation

or the ability of the cells to proliferate (data not shown).

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Figure 6:3 - Effect of blocking CD1 and MHCII on AN5 polar fraction driven cell - mediated responses. (A) Proliferation measured by 3H - Thy incorporation and (B) IFNγ production after stimulation with AN5 - derived polar

lipids in the presence of serial dilutions of anti - CD1 (red bars), anti - MHCII (blue bars) or isotype control (grey bars) monoclonal antibodies. Hatched bars indicate response with no antibody added. Each bar represents the mean of triplicate responses in 3 animals tested with the nil value subtracted (Δ); * p < 0.05, *** p < 0.001 using repeated

measures ANOVA with Dunnett’s multiple comparison test. Lipids used at 20 µg ml-1 for (A) 5 days or (B) 12 - 16 hours.

Measurement of the level of IFNγ produced in the same experiment is shown in figure 6:3

B. Again, a strong IFNγ response was seen to the AN5 - derived polar fraction in the

absence of any antibody (figure 6:3 B, hatched bar) and the addition of an isotype control

or the anti - CD1 monoclonal antibody had no effect on the levels of IFNγ produced

(figure 6:3 B, grey bars and red bars respectively). Addition of the anti - MHCII

monoclonal antibody did cause a small, dose dependant reduction in IFNγ production

(figure 6:3 B, blue bars). No antibody - mediated effect was seen with the background

level of IFNγ production or the ability of the cells to respond to PWM (data not shown).

Although blockade of MHCII appeared to reduce proliferative and cytokine responses it

was still unclear what sort of molecule might be driving these responses. To this end, the

polar lipid fraction was treated with Proteinase K to degrade any lipopeptide present in

the fraction. Further, to ensure that the heating required during Proteinase K treatment,

or the presence of inactivated Proteinase K itself, did not affect the responses, a mock

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treatment was also performed. Treatment of the AN5 polar fraction with Proteinase K

led to reduced proliferation in PBMC (figure 6:4) when compared to the untreated polar

fraction. Mock Proteinase K treatment did not alter the ability of the fraction to drive

strong proliferative responses (figure 6:4).

Figure 6:4 - Effect of Proteinase K treatment on proliferative ability of the AN5 polar lipid fraction. Proliferation measured by 3H - Thy incorporation after stimulation of PBMC with the AN5 polar lipid fraction. Each bar represents the mean of triplicate responses in 3 animals tested; ** p < 0.01 using Friedman repeated measures ANOVA

with Dunn’s multiple comparisons test.

Together these data demonstrate a large degree of MHCII restriction within the AN5 -

derived polar fraction and it is clear that some of the proliferative response driven by this

fraction can be degraded by treatment with Proteinase K. However, neither of these

methods completely abrogate the response suggesting that a lipid factor may be involved

in driving proliferation.

As the densitometry analysis performed on the AN5 polar fraction (figure 3:6) had

showed that over half of the total AN5 derived polar lipid fraction consisted of PIMs

(table 3:2), it was considered a possibility that the non - lipopeptide mediated responses

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seen could be due to PIM activity. To address this, 5 highly purified PIMs were obtained

through a collaborator and used to stimulate bovine PBMC.

Adaptive Immune Responses to Purified PIM Molecules

PBMC from 10 naturally M. bovis infected cattle were cultured for 5 days in the

presence of each PIM molecule and proliferative responses were measured. Both the

frequency and strength of proliferative responses differed depending on the nature of

the PIM molecule (figure 6:5 A). PIM2 failed to induce a proliferative response in any of

the animals studied, while AcPIM2 induced responses in only 3 out of 10 animals. In

contrast, Ac2PIM2, AcPIM6 and Ac2PIM6 were more frequently recognised, inducing

proliferative responses in 6 (Ac2PIM2 and AcPIM6) and 7 (Ac2PIM6) out of 10 animals.

Overall, significantly greater PBMC proliferation was detected in the Ac2PIM2, AcPIM6 and

Ac2PIM6 treatment groups compared to nil stimulated controls, with median values

tending to be greater following AcPIM6 stimulation (figure 6:5 A).

In addition to measuring proliferative responses, the ability of the PIM molecules to

induce IFNγ responses in the same animals was assessed (figure 6:5 B). Again, the

frequency of responding animals differed depending upon the nature of the PIM

molecule. AcPIM2 was least recognised, inducing responses in only 2 of the 10 animals.

PIM2, Ac2PIM2 and Ac2PIM6 were more frequently recognised, with responses detected in

3 (PIM2) and 4 (Ac2PIM2 and Ac2PIM6) out of 10 animals. AcPIM6 was most frequently

recognised, inducing responses in half of the animals studied. Furthermore, AcPIM6 was

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the only PIM molecule to induce significantly greater levels (p < 0.01) of IFNγ overall

when compared to nil stimulated controls (figure 6:5 B).

Figure 6:5 - Effect of stimulation with purified PIMs on bovine PBMC. (A) Proliferation measured by 3H - Thy incorporation; (B) IFNγ production measured by Bovigam. Points represent

triplicate wells for each of 10 animals tested; lines indicate sample median; * p < 0.05, ** p < 0.01, *** p < 0.001 using Friedman repeated measures ANOVA with Dunn’s multiple comparisons test.

Phenotyping of AcPIM6 Responsive Cells by Flow Cytometry

As AcPIM6 was the only PIM molecule to generate significantly increased levels of

IFN-γ (figure 6:5 A) and produced the greatest increase in the median proliferative

response (figure 6:5 B), this antigen was used to stimulate PBMC from an M. bovis

infected animal to characterise the proliferating cell populations by flow cytometry.

CellTrace Violet labelled cells were incubated for 5 days with antigen before being

harvested and labelled for flow cytometric analysis. After stimulation with either PPD - B

or AcPIM6, 3 populations of proliferating cells were identified based on cell surface

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phenotyping: (i) CD4+ T cells (CD3+ CD4+); (ii) CD8+ T cells (CD3+ CD8+); and (iii) NKT like

cells (CD3+ CD335+). An example of the gating strategy for identifying proliferating NKT

like cells is highlighted in figure 2:4, which demonstrates a greater level of proliferating

cells in response to stimulation with AcPIM6 (58.29 %) when compared to the nil antigen

control (29.87 %).

Figure 6:6 - Assessment of proliferating cell phenotype by flow cytometry Proliferation of CD3+ CD4+, CD3+ CD8+ or CD3+ CD335+ cells in response to either PPD - B (grey bars) or AcPIM6 (red

bars). Each bar represents the percentage of cells proliferating after subtraction of the unstimulated control.

The effect of stimulation with either PPD - B or AcPIM6 on the three different cell

populations are summarised in figure 6:6. Stimulation with PPD - B drove antigen specific

proliferation of approximately 60 % of the CD4+ T cells (CD3+ CD4+). Similarly, an antigen

specific proliferative response was seen in approximately 15 % of the CD8+ T cells (CD3+

CD8+) to PPD - B. A slight increase in NKT cell (CD3+ CD335+) proliferative responses

(approximately 20 %) was also seen to these antigens (figure 6:6).

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Stimulation with AcPIM6 induced only limited proliferation of CD4+ T cells (approximately

5 %) and no proliferation of CD8+ T cells above the background (figure 6:6). In contrast,

approximately 30 % of the NKT cell population mounted a proliferative response after

stimulation with AcPIM6 (figure 6:6). Little or no proliferation above the unstimulated

negative control was seen in the CD3- CD335+ cells or the CD3+ γδTCR+ populations (data

not shown).

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Discussion

Having tested the lipid subfractions on bovine MDDC and found no differences

(see Chapter Five), it appeared that both lipid and lipopeptide may have an important

role to play in the ability to mediate bovine immune responses. Lipopeptides have been

shown to be important immune mediators, primarily in the context of adaptive

immunity(257, 360, 364) and the fact that lipids can generate adaptive immune responses has

been well documented(199, 202, 205, 209, 215, 216, 226, 342, 350, 352, 353). However, before assessing

the potential roles of lipopeptide and lipid on bovine adaptive immune responses,

the existing lipid fractions had to be screened to see if they were capable of driving such

responses.

Similarly to the effects seen when screened in MDDC, the polar lipid fraction generated

significantly greater responses in PBMC than the apolar fraction (figure 6:1) and the

response driven by the AN5 - derived polar lipids was comparable or stronger (figure 6:2).

There is no difference in overall lipid content between the fractions when analysed by

TLC (figure 3:1 and figure 3:3), nor is any difference apparent after staining with ninhydrin

(figure 3:2 and figure 3:4). However, analysis of the densitometry of both fractions

highlighted the largest difference between the two bacterial strains is the proportion of

the polar fraction made up by PIM which may account for the stronger responses

generated after exposure to the AN5 - derived lipid fraction.

To assess the role of lipopeptide in the AN5 polar fraction, proliferation and IFNγ were

measured after either the addition of antibodies to block MHCII or CD1 or after treatment

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ofthe lipid fractions with Proteinase K. The blocking of MHCII, but not CD1, reduced

ability of the AN5 - derived polar fraction to drive proliferation (figure 6:3) as did

treatment of the fraction with Proteinase K (figure 6:4). Clearly a proportion of the

responses measured are restricted through MHCII and degradable by Proteinase K

treatment, but these treatments do not completely abrogate the responses. It may be

that the remaining responses are lipid mediated, however the possibility that some

lipopeptide remains after Proteinase K treatment or that MHCII blocking is not absolute

cannot be ignored. These data are in line with other work showing that Proteinase K

treatment of chloroform : methanol extracts of M. tuberculosis reduced, but did not

abrogate, the ability of the fraction to drive IFNγ production from human PBMC(257).

Interestingly, the authors also treated their fraction with lipase and found similar results,

suggesting that, in crude extracts at least, lipopeptide plays a role in driving T cell

responses but is not responsible for all the activity seen. More recent studies have

highlighted specific mycobacterial lipopeptides capable of modulating the CD4+ T cell

response(364) and suggested a recognition mechanism(360).

Many cell populations found within PBMC have been shown to be affected by lipid

antigens. Invariant NKT cells, restricted through CD1d, are particularly well

characterised(356, 357) but conventional CD4+ T cells have been shown to be reactive to

lipids presented in the context of CD1b(202, 203) and lipid - induced, IL - 17 secreting γδ TCR+

T cells have been shown to be important in early granuloma formation(359).

Unfortunately, the presence of lipopeptide in the crude fractions, as well as the

subfractions, meant that they were not suitable for further dissection of the bovine

adaptive immune response to lipids. To address this, a selection of highly purified PIM

136


molecules was obtained and the ability of these highly purified PIMs to drive immune

responses was assessed.

The larger and more complex PIM molecules tested (Ac2PIM2, Ac2PIM6 and AcPIM6) drove

significant levels of proliferation (figure 6:5 A) while only AcPIM6 was able to drive

significant production of IFNγ (figure 6:5 B). Interestingly, the same antigens were used

to stimulate whole blood overnight and the supernatants were assayed for IFNγ but none

was seen (data not shown).

There are very few studies showing the effect of mycobacterial lipids in short incubation,

whole blood assays. Cell - mediated immune responses to lipid antigens are more

commonly assessed by EliSPOT with incubation times of at least 48 hours required before

measurable responses become apparent(228, 300). The discrepancy between the whole

blood and PBMC IFNγ responses may be due in part to the requirement for antigen

processing and presentation of PIMs(208). Another possibility is that the frequency of lipid

responsive cells is low and an extended incubation allows for expansion of these cells.

This is supported by the demonstration of strong proliferative responses induced after

stimulation of PBMC with PIMs (figure 6:5 A). A recent study has shown that bovine NKT

cells are present only at low frequencies in peripheral blood (0.1 % - 1.7 %)(365).

Previous work using mice has shown that the ability of PIMs to generate cell - mediated

responses is dependent on the acyl structures of the molecules. Early work performed

using PIM2 and PIM6 demonstrated that the acyl chain was essential for NKT cell

recruitment while the complexity of the mannose residues did not alter the response(323)

137


however it was subsequently shown that the second acyl chain of PIM4 enhances binding

to murine CD1d but that the polar mannose head was essential for antigen recognition,

proliferation and IFNγ production(228). As well as the number and location of acyl chains,

their degree of unsaturation and cis, but not trans, stereochemistry is critical in

determining antigenicity(357, 366).

As the only molecule to drive significant IFNγ responses and one of the most potent

inducers of proliferation, AcPIM6 was used to characterise the proliferative response.

Stimulation with AcPIM6 induced higher levels of proliferation in NKT cells than in CD4+ or

CD8+ T cells (figure 6:6). However, from these data it is not possible to tell if the

proliferative CD4+ or CD8+ also co - express CD335 as our flow cytometric labelling panels

do not allow the discrimination, however this is a distinct possibility.

Although well characterised in humans and mice, the presence of NKT cells in cattle has

been a controversial issue. Originally bovine CD1D was identified as a pseudogene(367, 368)

and it was assumed that cattle lacked an NKT population as CD1D genes are a

prerequisite for NKT cell development(369, 370). Nevertheless, further studies have shown

that despite its unusual structure the bovine CD1D gene is expressed and translated in

vivo(371). Furthermore, recent work has identified a subset of cattle lymphocytes that

express both T cell (CD3) and NK cell (NKp46) markers, suggesting the presence of an NKT

cell population in bovine peripheral blood(365). In addition to CD1d restricted invariant

cells, human CD1b restricted variant NKT cells have been described in transgenic mice(355)

and, given the presence of functional CD1b in cattle, the existence of a CD1b restricted

NKT cell population cannot be ruled out. Furthermore, bovine NKT cells have been

138


shown to express both αβ and γδ TCRs, have a broad TCR repertoire and have fully

functional NKp46, CD16 and CD3 signalling pathways(365). Interestingly these cells require

ligation of their CD3 to produce IFNγ. While this may initially suggest a CD3 binding

component may be present in the PIM preparations, it is worth noting that the cytokine

producing cells have not been identified.

In conclusion, this chapter demonstrates that the crude lipid preparations are capable of

generating adaptive immune responses. A significant portion of the proliferative

response was shown to be restricted through MHCII and treatment of the lipid fraction

with Proteinase K also reduced its proliferative activity. The use of highly purified PIM

molecules enabled phenotypic identification of NKT proliferating in response to PIM

stimulation. Given that NKT cells have been shown to be important in the development

of protective immunity to certain intracellular pathogens including the influenza virus(372)

and Leishmania major(373), lipid molecules such as AcPIM6 may have a role to play in

future vaccines either as protective antigens or biological adjuvants.

139


Chapter Summary

• Objective

Assessment of the lipid fractions ability to generate adaptive immune

responses and identify if the presence of lipopeptide in the fractions was

responsible for any stimulation. Characterisation of responding cell

populations within PBMC.

• Results

Crude polar lipid fractions were capable of generating both proliferative and

cytokine responses. Much of the proliferative response was shown to be

restricted through MHCII and the proliferative activity of the polar fraction

could also be reduced by treatment with Proteinase K. Not all proliferative

response was driven through MHCII or degraded by Proteinase K activity

suggesting a lipid component existed. As the most abundant lipid family in the

polar fraction, highly purified PIMs were used to stimulate PBMC and NKT

were cells found to be the main proliferative population.

• Conclusions

The presence of lipopeptide in lipid preparations is of significance and may be

responsible for much of the response seen when using crude fractions.

However, lipid molecules are also capable of driving functional responses from

cells of the adaptive immune system.

Bovine NKT cells are capable of recognising, and proliferating in response to

the highily mannosylated lipid AcPIM6.

140

Chapter Seven Concluding Remarks

Chapter Seven

Concluding Remarks

The primary purpose of this study has been to address the hypothesis that lipids

from M. bovis constitute a source of molecules that could aid in the development of

control measures for BTB such as subunit vaccines (as antigens or immunomodulatory

adjuvants) or diagnostic reagents. To work towards these goals it is necessary to

characterise their immunological properties and to identify individual compounds with

the desired properties. The data presented here describe bovine immune responses

directed at lipids extracted from 2 strains of M. bovis.

The 3 main objectives of the work reported here were laid out at the end of Chapter One

and each shall be addressed here:

• Develop methods to isolate and characterise lipid moieties from M. bovis:

While the method used to extract polar and apolar lipid fractions in this study is

not novel, a full 2D TLC analysis of lipids from M. bovis AF 2122/97 has not been

published previously. At this macroscopic level, little difference can be seen

between AF 2122/97 - derived and AN5 - derived lipid fractions. However, the

application of densitometrical analysis allowed for some further refinement to the

comparisons. Also shown here, and not previously published, is ninhydrin staining

141


of these lipid fractions which demonstrated the presence of a range of

unidentified amine residues within the polar fractions. This work constitutes the

first published documentation of the polar and apolar lipid fractions from M. bovis

AF 2122/97.

Further analysis of the polar fraction was attempted by chemical subfractionation.

This proved to be technically demanding, partly due the poor efficiency of column

chromatography methods and subsequently due to the difficulty in removing

colloidal silica from TLC preparations. Despite these challenges, lipid subfractions

were successfully prepared but ninhydrin staining of 2D TLCs demonstrated

lipopeptide contamination in all subfractions.

That lipopeptides can mediate immune responses is well known but, despite this,

no comprehensive lipopeptide screening has been published for any members of

the M. tuberculosis complex. The ability to extract and purify lipids using simple

methods is of limited use without further work to analyse and identify lipopeptide

contaminants and their role in modulating host immunity.

• Assess the effect of M bovis lipids on bovine APC to identify potential

immunomodulatory activity which could be exploited in the development of

novel adjuvants:

Stimulation of bovine MDDC with the polar and apolar lipid fractions showed that

components within the polar lipid fraction are capable of mediating effects on the

host APC. Significant reduction in important molecules such as MHCII and CD1b

was seen, as well as increased IL - 10 and IL - 12 secretion, and these responses

could hamper the hosts ability to mount an appropriate immune response.

142


Despite the presence of lipids known to stimulate Th1 responses, the potentially

deleterious effect of the polar lipids on APC may constitute a strategy employed

by the bacilli to increase the chances of successful infection. Further refinement

of the polar fraction was attempted to try to identify which moieties might be

responsible for these effects however, despite the different lipid composition of

the subfractions, no differences were found in the ability of the subfractions to

mediate reduction in MHCII expression or increase the secretion of IL - 10.

Despite the increased levels of IL - 12 measured after stimulation of APC with the

polar lipids, the elevated levels of IL - 10 and reduction in important antigen

presentation molecules mediated by the lipid fractions is precisely the opposite of

an adjuvanting response.

Clearly further work is required on purification of individual lipids. More complex

chemical technologies and more advanced purification methods have been

applied by others to obtain individual lipid molecules free from contamination

with lipopeptide and it is these approaches that must be taken to further separate

and define the lipid fractions from M. bovis.

• Identify the lipid targets of the bovine adaptive immune response which could

be used as innovative vaccine candidates:

Initial experiments documented in this thesis demonstrated the ability of the

crude lipid fractions to drive proliferative and cytokine responses in bovine PBMC.

Interestingly, stronger responses were seen to the polar fraction, as had been the

case with innate immune cells. The finding that much of the proliferative

response to the polar fraction was restricted through MHCII and could be partially

143


degraded by treatment of the lipid fraction with Proteinase K again highlights the

potential role of lipopeptide in these lipid preparations. However, neither

blockade of MHCII nor enzymatic degradation of lipopeptide completely

abrogated proliferative responses and the use of purified PIM molecules enabled

the identification of AcPIM6 as a natural ligand of bovine NKT cells. The presence

of these cells in cattle has only recently been shown and the discovery of a ligand

so soon is a remarkable achievement.

Whether or not AcPIM6 or other lipid molecules could be used as vaccine

candidates remains an open question. The non - polymorphic nature of CD1

molecules makes them good targets for a vaccine antigens and the ability to

target a specific cell population with an individual antigen has been demonstrated

in this thesis. However, the restriction of the proliferating NKT cells is currently

unknown but warrants further investigation.

The main achievements of this thesis are the extraction and screening of the M. bovis -

derived lipid fractions, the documentation of the effects of the lipid fractions on bovine

APC and the identification of a specific lipid ligand capable of driving proliferation in

bovine NKT cells. However, this thesis also highlights the lack of knowledge surrounding

the lipopeptide content of the mycobacterial cell wall and underlines the requirement for

cross - discipline collaboration to separate the individual components of these extracts

and fully understand the interaction at the point of contact between host and pathogen.

144

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Appendix

Appendix

Appendix

Publications Associated with this Thesis

C. Pirson, G. J. Jones, S. Steinbach, G. S. Besra & H. M. Vordermeier, (2012). “Differential

effects of Mycobacterium bovis - derived polar and apolar lipid fractions on bovine

innate immune cells” Veterinary Research 43:54

C. Pirson, R. Engel, G. J. Jones, T. Holder, O. Holst & H. M. Vordermeier, (2015) “Highly

purified mycobacterial phosphatidylinositol mannosides drive cell mediated responses

and activate NKT cells in cattle” Clinical and Vaccine Immunology 22:2

Poster presented at VIth International M. bovis Conference

Differential effects of Mycobacterium bovis - derived polar and apolar lipid fractions on

bovine innate immune cells

217

VETERINARY RESEARCHPirson et al. Veterinary Research 2012, 43:54http://www.veterinaryresearch.org/content/43/1/54

RESEARCH Open Access

Differential effects of Mycobacterium bovis -derived polar and apolar lipid fractions on bovineinnate immune cellsChris Pirson1*, Gareth J Jones1, Sabine Steinbach1, Gurdyal S Besra2 and H Martin Vordermeier1

Abstract

Mycobacterial lipids have long been known to modulate the function of a variety of cells of the innate immunesystem. Here, we report the extraction and characterisation of polar and apolar free lipids from Mycobacterium bovisAF 2122/97 and identify the major lipids present in these fractions. Lipids found included trehalose dimycolate(TDM) and trehalose monomycolate (TMM), the apolar phthiocerol dimycocersates (PDIMs), triacyl glycerol (TAG),pentacyl trehalose (PAT), phenolic glycolipid (PGL), and mono-mycolyl glycerol (MMG). Polar lipids identifiedincluded glucose monomycolate (GMM), diphosphatidyl glycerol (DPG), phenylethanolamine (PE) and a range ofmono- and di-acylated phosphatidyl inositol mannosides (PIMs). These lipid fractions are capable of altering thecytokine profile produced by fresh and cultured bovine monocytes as well as monocyte derived dendritic cells.Significant increases in the production of IL-10, IL-12, MIP-1β, TNFα and IL-6 were seen after exposure of antigenpresenting cells to the polar lipid fraction. Phenotypic characterisation of the cells was performed by flowcytometry and significant decreases in the expression of MHCII, CD86 and CD1b were found after exposure to thepolar lipid fraction. Polar lipids also significantly increased the levels of CD40 expressed by monocytes and culturedmonocytes but no effect was seen on the constitutively high expression of CD40 on MDDC or on the levels ofCD80 expressed by any of the cells. Finally, the capacity of polar fraction treated cells to stimulate alloreactivelymphocytes was assessed. Significant reduction in proliferative activity was seen after stimulation of PBMC by polarfraction treated cultured monocytes whilst no effect was seen after lipid treatment of MDDC. These datademonstrate that pathogenic mycobacterial polar lipids may significantly hamper the ability of the host APCs toinduce an appropriate immune response to an invading pathogen.

IntroductionBovine tuberculosis (BTB), caused by Mycobacteriumbovis (M. bovis), is a zoonotic disease of significant eco-nomic, animal and public health burden. Consumptionof raw or unpasteurised animal products and contactwith infected carcasses plays a large role in zoonotic M.bovis infection of humans in Africa and South America[1,2]. Yet this is not a problem solely associated withpoverty and less developed countries as evidenced by arecent report that up to 45% of all TB infected childrenin San Diego were caused by M. bovis [3]. The incidenceof BTB in cattle in Great Britain (GB) has undergonesteady and continual increase since 1998 despite the

* Correspondence: [email protected] Research Group, Animal Health and Veterinary Laboratories Agency –Weybridge, New Haw, Surrey, Addlestone KT15 3NB, United KingdomFull list of author information is available at the end of the article

© 2012 Pirson et al.; licensee BioMed Central LCommons Attribution License (http://creativecreproduction in any medium, provided the or

implementation of control measures, possibly due to thepresence of a wildlife reservoir [4]. Within GB, BTB hasspread drastically since the Foot & Mouth disease out-break in 2001 with the annual number of animalsslaughtered rising from a mean of 7116 animals between1998 and 2001 to an annual mean of 26 277 cases be-tween 2002 and 2010 inclusive [5].The first point of contact between M. bovis and its

host is likely to be interaction between receptors of itssentinel cells of the innate immune system such asmacrophages or dendritic cells (DC) and the surfaceexpressed molecules of the bacilli, the majority of whichare lipid in nature [6,7]. Recognition of mycobacteriallipids by macrophages has been demonstrated via themannose receptor, complement receptors, scavengerreceptors and CD14 [8-10]. Furthermore, various lipids

td. This is an Open Access article distributed under the terms of the Creativeommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andiginal work is properly cited.

mailto:[email protected]

http://creativecommons.org/licenses/by/2.0

Pirson et al. Veterinary Research 2012, 43:54 Page 2 of 11http://www.veterinaryresearch.org/content/43/1/54

from M. tuberculosis have been shown to ligate TLR2 onhuman macrophages [11]. DC are known to possess andutilise these same receptors as well as other, structurallyrelated molecules such as the DC specific C - type lectin(DC - SIGN) which have also been heavily implicated inimmune recognition of the bacilli [12-14].Many lipids have been implicated in mycobacterial

virulence which are not found in other bacterial genera.Lipomannan (LM), lipoarabinomannan (LAM), thephosphatidylinositol mannosides (PIMs), the cord fac-tors trehalose mono- and dimycolate (TMM and TDM)and the phthiocerol dimycocerosates (PDIMs) are allsurface bound mycobacterial lipids capable of modulat-ing innate immunity [15-17]. Recently, newly identifiedlipids, such as monomycolyl glycerol (MMG), have beenshown to modulate host immunity [18] and hyperviru-lence [19]. However, little data exist which describe theeffect of M. bovis derived lipid on bovine innate cells.Previously published work usually relies on non - patho-genic mycobacterial species or makes use of an animalmodel rather than the pathogen’s natural host. For ex-ample, work performed by Hope et al. uses bovinemonocyte-derived DC (MDDC) but these cells are sti-mulated with a synthetic lipopeptide [20] rather than aM. bovis-specific lipid antigen. Additionally, althoughReed et al. demonstrated that blockage of synthesis ofthe phenolic glycolipid (PGL) correlated with increasedsecretion of TNF-α, IL - 6 and IL - 12 by the host, andremoved the “hyperlethal” phenotype displayed by thebacilli, this was only shown in the murine model [19].Thus, to assess host immune responses to its natural

pathogen’s lipid constituents, lipids were extracted fromvirulent M. bovis AF 2122/97 and used to stimulate vari-ous bovine innate immune cells isolated from live, TBfree, cattle. Cellular responses were evaluated by measur-ing cytokine production, alterations in cell surface mole-cules and induction of T-cell proliferation.

Materials and methodsPreparation of bacterial isolates for lipid extractionBacterial isolates were grown in Middlebrooks 7H9medium as previously described [21].Briefly, bacterial cells were grown in 100 mL volumes

in rolling culture flasks inoculated with 1 mL of a startingculture. At mid - log phase, cultures were decanted intosterile tubes and pelleted before being washed twice insterile water. Finally, pellets were resuspended in 5 mLsterile water and heat killed in a water bath at 80 °C to90 °C for between 1 and 2 h and finally freeze dried.

Extraction of crude free mycobacterial lipidThe extraction of mycobacterial lipid has been previ-ously described [22]. Briefly, freeze dried bacterial cellswere suspended in methanolic saline before an equal

volume of petroleum ether was added and the mixturestirred for 12 to 16 h. Cells were pelleted by centrifuga-tion (7000 g for 10 min) and the non-aqueous phasecontaining the apolar lipids was removed and stored. Anequal amount of petroleum ether was added to the aque-ous lower phase and the mixture stirred for 2 h beforebeing centrifuged (7000 g for 10 min) and the non-aqueous layer removed and pooled with the first. Thesenon-aqueous petroleum ether extracts were dried using arotary evaporator with cold finger condenser and thelipid transferred to a pre-weighed glass tube in 4 : 1CHCl3 : CH3OH. Evaporation of the CHCl3 : CH3OHwas achieved using a heating block and N2 gas streamand the tube weighed to determine the mass of apolarlipids extracted.Extraction of polar lipids was performed by adding

CHCl3, CH3OH and 0.3% aqueous NaCl in a 9 : 10 : 3ratio to the cell pellet. The mixture was stirred for 12 to16 h before being passed through 2 Whatman # 91 fil-ters. Once dried, the cells were recovered from the filterpapers and re-extracted twice using a 5 : 10 : 4 mixtureof CHCl3, CH3OH and 0.3% NaCl. After a final filtrationto remove the cells, equal volumes of CHCl3 and 0.3%NaCl were added and the mixture stirred for one hour,after which the aqueous phase containing the polarlipids was removed and dried in a rotary evaporator.Final polar lipid mass was ascertained as described forthe apolar petroleum ether extracted lipid fraction.

Analysis of lipid fractions by 2D Thin LayerChromatography (TLC)Aluminium backed silica gel 60 F254 TLC plates (FisherScientific, Loughborough, Leics, UK) were cut into ap-proximately 6 cm squares and 100 μg of lipid extractwas spotted onto the plates using glass micro - capillarypipettes. Plates were dried thoroughly before beingplaced in TLC tanks containing appropriate solvent mix-tures (systems A - E, Table 1). TLC plates were dried be-tween each run and before staining to ensure thatresidual solvent was removed. Staining was performedusing a 5% solution of molybdophosphoric acid (MPA)(Sigma Aldrich, Poole, Dorset, UK) in 95% ethanol (Fig-ure 1). Stains were sprayed onto TLC plates which weresubsequently charred using a hot air gun before beingphotographed or scanned. Identification of individuallipids was performed by comparison with previouslypublished TLC analysis [23].

CattleBTB free cattle between the ages of 6 and 36 monthswere obtained from herds within 4 - yearly testingparishes with no history of a BTB breakdown in the past4 years. These animals were purchased when around6 months old and transported to AHVLA. Whilst at

Table 1 Solvent systems for TLC analysis of mycobacterial lipids (adapted from [22]).

Solvent System Run Direction Components Runs Lipids Resolved

A 1 petroleum ether : ethyl acetate (98 : 2) 3 PDIM, TAG, MQ

2 petroleum ether : acetone (98 : 2) 1

B 1 petroleum ether : acetone (92 : 8) 3 AT, FA

2 toluene : acetone (95 : 5) 1

C 1 chloroform : methanol (96 : 4) 1 FA, GLY

2 toluene : acetone (80 : 20) 1

D 1 chloroform : methanol : water (100 : 14 : 0.8) 1 CF, SL, DAT

2 chloroform : acetone : methanol : water (50 : 60: 2.5 : 3) 1

E 1 chloroform : methanol : water (60 : 30 : 6) 1 DPG, PE PI, PIM

2 chloroform : acetic acid : methanol : water (40 : 25 : 3 : 6) 1


AHVLA they tested negative for BTB using both theBovigam IFNγ assay (Prionics AG, Schlieren-Zurich,Switzerland) and the single intradermal comparative cer-vical tuberculin test (SICCT).

Isolation of bovine PBMC from whole bloodWhole blood was mixed in equal amounts with sterileHBSS containing 10 U mL-1 heparin. This mixture wasoverlaid onto Histopaque 1077 (Sigma Aldrich) and cen-trifuged at 800 g for 40 min. The PBMC interface wasremoved using a pastette and washed twice in HBSScontaining heparin. Live cells were identified via trypanblue exclusion and enumerated using a haemocytometer.

Isolation of CD14+ monocytes from bovine PBMCPBMC were suspended in 80 μL of MACS rinsing bufferper 107 cells before the addition of 10 μL of MACS anti- CD14 MicroBeads (Miltenyi Biotec, Bisley, Surrey, UK)per 107 cells. After a 15 min incubation at +4 °C on a ro-tator, cells were pelleted and resuspended in 500 μL per108 cells and passed through MACS LS columns as perthe manufacturer’s instructions.The CD14+ fraction was counted and cells diluted to

1.5 × 106 mL-1 in cell culture medium (RPMI 1640containing 25 mM HEPES, 10% FCS, 1% NEAA,5 × 10-5 mM β2 - mercapto-ethanol, 100 U mL-1 penicil-lin and 100 μg mL-1 streptomycin [Gibco Life Technolo-gies, Paisley, UK]).

Generation of bovine cultured monocytes and MDDCCD14+ monocytes were plated in 1 mL volumes at1.5 × 106 mL-1 in 24 well plates (Nunc Nunclon, Ros-kilde, Denmark) before adding either 1000 U mL-1

equine GM-CSF (Kingfisher Biotech, St Paul, MN, USA)(cultured monocytes) or 1000 U mL-1 equine GM-CSFand 4 ng mL-1 bovine IL-4 (AbD-Serotec, Kidlington,Oxon, UK ) (MDDC). Cells were cultured at 37 °C+ 5%CO2 for 3 days [20], following which they were har-vested, re-plated at 1.5 × 106 mL-1 in fresh cell culture

medium and the appropriate volume of lipid solutionwas added. Cells were cultured for a further 12 to 16 hbefore supernatants were collected and cells harvestedfor subsequent flow cytometric analysis.

Preparation of lipid antigen suspensionsSuspensions of all lipid antigens were prepared in anaqueous phase for use in cell culture experiments afterfirst removing any CHCl3 : CH3OH by evaporation usingan N2 gas stream. Cell culture medium was added to thedried lipid and the mixture subjected to 2 cycles of heat-ing at 80 °C and then sonication for 5 min. Apolar andpolar lipids were used to stimulate cells in vitro at20 μg mL-1 for 12 to 16 h. These conditions were shownto be optimal in previous experiments (data not shown).

Measurement of cytokine productionCulture supernatants were assayed for cytokine levelsusing the MSD multiplex platform (Meso Scale Discov-ery, Gaithersburg, MD, USA) as previously described[24,25]. Briefly, supernatants were analysed using a cus-tom multiplex electrochemiluminescent system whichallows simultaneous detection of IL-1β, IL-6, IL-10,IL-12, MIP-1β and TNF-α (Meso Scale Discovery).Multiplex 96 well plates were supplied with target cap-ture antibodies spotted onto 6 separate carbon electro-des in each well (anti-bovine TNF-α [Endogen,Rockford, IL, USA]; anti-bovine IL-10 and anti-bovineIL-12 [AbD-Serotec]; anti-bovine IL-1β, anti-bovine IL-6and cross-reactive anti-human MIP-1β [Meso Scale Dis-covery]). Plates were blocked with MSD assay buffer for30 min at room temperature before the addition of sam-ples or standards for 1 h at room temperature. Recom-binant standard controls (Meso Scale Discovery) wereprepared by serial dilution. After incubation, plates werewashed and combined biotinylated secondary detectorantibodies were added for a further hour. Finally, plateswere washed, loaded with MSD read buffer and analysedusing an MSD Sector Imager 6000.

Figure 1 2D TLC analysis of crude, free lipids extracted from M. bovis AF 2122/97 and stained with MPA. A - D: Apolar fraction analysedwith TLC systems A, B, C and D. E and F: Polar fraction analysed with systems D and E. PDIM - phthiocerol dimycocerosate; MQ - menaquinone;TAG - triacyl glycerol; PAT - pentacyl trehalose; PGL - phenolic glycolipid; MMG - monomycolyl glycerol; TMM - trehalose monomycolate; TDM -trehalose dimycolate; GMM - glucose monomycolate; DPG - diphosphatidyl glycerol; PE - phosphatidyl ethanolamine; PIM - phosphatidylinositolmannosides (integers denote number of mannoside or acyl groups); PI - phosphatidyl inositol; P - phospholipid.


Cell labelling and analysis by flow cytometryCultured cells were suspended in PBS and labelled withthe live / dead indicator ViViD (Invitrogen Life Tech-nologies, Paisley, UK) before being transferred to a 96

well plate and washed using 150 μL MACS rinse buffer.Cells were stained for 15 min using either anti-bovineCD14 (ccG33; Institute for Animal Health; 1:50 dilu-tion), anti-equine MHCII (MCA1085; AbD Serotec; 1:50


dilution), anti-bovine CD40 (IL-A156; cell supernatant;AHVLA; 1:10 dilution), anti-bovine CD80 (IL - A159;cell supernatant; AHVLA; 1:10 dilution), anti-bovineCD86 (IL-A190; cell supernatant; AHVLA; 1:10 dilu-tion), anti-bovine CD1b (CC14; AbD Serotec MCA831G;1:10 dilution) or an IgG1 isotype control (Av20; Institutefor Animal Health; 1:50 dilution). Labelled cells werewashed using 150 μL MACS rinse buffer and secondarylabelling was performed using a 1:400 dilution of anti-IgG1 conjugated to R-Phycoerythrin (R-PE) (Invitrogen;P21129) in 50 μL volumes for 10 min. After incubation,cells were washed by the addition of 150 μL PBS, pel-leted and resuspended in 100 μL of 2% paraformalde-hyde (Cytofix; BD Biosciences, Oxford, Oxon, UK) for atleast 30 min at 4 °C before analysis on a CyAn ADP ana-lyser. For capture and analysis, initial gating was on sin-gle, ViViDlo (live) cells into a subsequent small cell/lymphocyte exclusion gate.

One way mixed lymphocyte reactionBovine MDDC and cultured monocytes were preparedfrom 1 animal as described above. Following 3 days inculture, cells were pulsed with lipid antigen overnightbefore being enumerated, washed to remove any cyto-kines and lipid from the media and incubated at 107

cells mL-1 in the presence of Mitomycin C at100 μg mL-1 for 30 min at 37 °C + 5% CO2. Lipid pulsed,Mitomycin C treated MDDC or cultured monocyteswere cultured in 1 mL at 37 °C + 5% CO2 at 2 × 105 with1 × 105 PBMC isolated from a second, allogeneic animal.After 5 days, cells were pulsed overnight with 1 μCiwell-1 of 3H-thymidine before being harvested using aHarvester 96 Mach III (TomTec Inc, Hamden, CT,USA). Lymphocyte proliferation was assessed by theincreased cellular incorporation of 3H-thymidine whichwas measured using a MicroBeta2 2450 (Perkin Elmer,Waltham, MA, USA).

Data and statistical analysisAll data representation and statistical analysis was per-formed using GraphPad Prism version 5.04 and Graph-Pad InStat version 3.06 (GraphPad Software, La Jolla,CA, USA). Flow cytometric data was analysed usingrepeated measures ANOVA with a Bonferroni multiplecomparisons post test. Cytokine profile analysis was per-formed using a Friedman non-parametric repeated mea-sures ANOVA with Dunns multiple comparisons test.

ResultsExtraction and analysis of lipid from AF 2122/97In order to identify individual lipid components withinthe polar and apolar fractions, lipids extracted from theM. bovis reference strain (AF 2122/97) were subjected to2D TLC analysis using solvent systems of increasing

polarity and subsequently stained with MPA. Analysis ofthe apolar fraction using the least polar TLC system(Figure 1a) identified the presence of phthiocerol dimy-cocerosates (PDIMs), menaquinone (MQ) and triacylglycerol (TAG). System B (Figure 1b) revealed that theapolar fraction also contained pentacyl trehalose (PAT),phenolic glycolipids (PGL) and monomycolyl glycerol(MMG). System C (Figure 1c) allowed further resolutionof both MMG and PGL. The most polar system used toanalyse the apolar fraction (system D, Figure 1d) identi-fied both trehalose monomycolate (TMM) and dimyco-late (TDM; cord factor) as well as glucose monomycolate(GMM).TLC analysis of the polar lipid fraction using system D

showed the presence of TMM and TDM (Figure 1e)while the most polar solvent system (E, Figure 1f )enabled identification of the most polar lipids, whichincluded diphosphatidyl glycerol (DPG), phosphatidylethanolamine (PE), phosphatidylinositol mannosides(PIMs, integers denote number of mannoside or acylgroups), phosphatidyl inositol (PI) and an unknownphospholipid (P).

Cytokine responses to M. bovis - derived lipidsInnate immune cells are known to produce cytokines inresponse to appropriate antigenic stimuli. In order to as-sess the effect of M. bovis - derived lipids on 3 types ofbovine innate cells (freshly isolated monocytes, culturedmonocytes and MDDC), cytokine production was mea-sured after stimulation with the lipid fractions (Figure 2).The cytokines investigated included IL-10, IL-12, TNF-α,MIP-1β, and IL-6.Significantly increased IL-10 secretion was seen from

all 3 cell types following stimulation with the polar lipidfraction (Figure 2a). Strong IL-10 responses were seenfor 3 animals, while only modest increases were notedfor the remaining cattle (Figure 2a). In contrast, little orno significant increase in IL-10 production was seen fol-lowing stimulation with the apolar lipid fraction, al-though apolar lipids induced some IL-10 production bycultured monocytes and MDDC from 3 animals. Allresponses to apolar lipids were by far lower than thoseinduced by the polar lipid fraction.IL-12 levels in the culture supernatants were measured

simultaneously and the results are shown in Figure 2b.Stimulation with the polar lipid fraction induced a largeincrease in IL-12 production by MDDC, while lowerresponses were also observed from monocyte and cul-tured monocyte populations. In contrast to the polarlipids, stimulation with the apolar lipid fraction resultedin minimal increases in IL-12 production by monocytes,cultured monocytes or MDDC.Levels of MIP-1β were also found to be significantly

increased after exposure to the polar lipid fraction with

Figure 2 IL - 10 (A), IL - 12 (B), MIP - 1β (C), TNFα (D) & IL - 6 (E) production by monocytes and cultured cells in response tostimulation with crude lipid fractions. Points represent mean responses from duplicate wells for each of 7 animals tested. Lines indicate thatcells were derived from the same animal; * 0< 0.05; ** p< 0.01; *** p< 0.001.


no significant increase seen after apolar lipid stimulation(Figure 2c). Both cultured monocytes and MDDC pro-duced noticeably more MIP-1β than fresh monocytes,with MDDC from 6, and cultured monocytes from 3, ofthe 7 animals responding strongly (Figure 2c).Significant increases in TNFα production were also

seen, again in response to the polar lipid fraction(Figure 2d). While polar lipid treated cultured mono-cytes from all 7 cattle produced significant levels of

TNFα, considerably more TNFα was produced byMDDC (Figure 2d). Further, the level of TNFα produc-tion was similar between fresh and cultured monocytes(Figure 2d).The production of IL-6 (Figure 2e) followed a broadly

similar pattern to that of TNFα (Figure 2d) althoughstatistical significance was not achieved. Fresh mono-cytes from 2 cattle produced more IL-6 after exposureto the polar lipids, which also drove increased IL-6

Figure 3 Surface expression of MHCII (A), CD86 (B), CD1b (C) and CD40 (D) on cultured cells and fresh monocytes after exposure tocrude polar and apolar lipid fractions. A single point represents the median fluorescence intensity of the specific stain after subtraction of anisotype control (ΔMFI) for each of 7 animals tested; ** p< 0.01; *** p< 0.001.


production in cultured monocytes from 6 cattle(Figure 2e). Polar lipid driven IL-6 production by MDDCwas noted in 5 of the 7 animals screened, with one ofthese animals producing more IL-6 to the apolar lipidfraction than the polar. While statistical significance wasnot achieved, the levels of IL-6 produced by MDDC arenotably higher than from fresh or cultured monocytes(Figure 2e).These data clearly demonstrate that the polar lipid

fraction drives the production of significant amounts ofIL-10, IL-12, MIP-1β and TNFα from all cell types. Fur-thermore, it is clear that MDDC produced more IL-12,TNFα and IL-6 than fresh or cultured monocytes andMIP-1β production is greater from both cultured mono-cytes and MDDC than in fresh monocytes.

Phenotypic responses to M. bovis - derived lipidsExposure of bovine antigen presenting cells to the polarlipid fraction lead to significant increases in the produc-tion of a variety of cytokines (Figure 2a-e), all of whichcan play important roles in directing the subsequent cellmediated response. In order to further assess the effectof M. bovis - derived lipids and the local cytokine milieuon these cells, analysis of the expression of key antigenpresentation related molecules was assessed by flowcytometry (Figure 3).

Stimulation with the polar lipid fraction resulted in asignificant reduction in the cell surface expression ofMHCII on all three cell types (Figure 3a). Furthermore,MHCII expression was also lower on MDDC followingstimulation with apolar lipids, although this did notachieve statistical significance (Figure 3a). CD86 expres-sion was also significantly reduced on all three cell typesfollowing stimulation with the polar lipid fraction(Figure 3b). While there was a trend for lower CD86 ex-pression on all three cell types following stimulationwith the apolar lipid fraction, this again did not achievestatistical significance (Figure 3b). Interestingly, thelipid-specific antigen presentation molecule CD1b,which was constitutively expressed on MDDC, was notpresent on monocytes or cultured monocytes nor couldit’s expression be modulated in these cell types by myco-bacterial lipids (Figure 3c). By contrast, MDDCexpressed CD1b constitutively and incubation with thepolar lipid fraction resulted in a significant reduction inCD1b surface expression (Figure 3c).Not all cell surface molecules were down-regulated

following treatment with lipids. CD40 expression onboth monocytes and cultured monocytes increased sig-nificantly following stimulation with the polar lipid frac-tion (Figure 3d), although no effect was seen on MDDC.Finally, no significant difference was seen in CD80 levels


following stimulation with either the polar or apolarlipid fractions (data not shown).In summary, these data demonstrate that M. bovis -

derived lipids, and in particular the polar fraction,downregulate the expression of several key cell surfacemolecules involved in antigen presentation.

Consequence of exposure to M. bovis - derived lipidsTo identify and assess any functional consequence of thelipid induced reduction in molecules related to antigenpresentation the ability of lipid treated innate cell typesto stimulate an alloreactive response was assessed. Co-culture of the responder PBMC population with eitheruntreated MDDC or cultured monocytes resulted in a 5- fold increase in their proliferation (Figure 4). No prolif-eration was noted for either MDDC or cultured mono-cytes in the absence of responder cells (data not shown).Polar lipid treated MDDC retained their ability to induceproliferation in the responder population despite thedownregulation of important costimulatory molecules.In contrast, allo - stimulation of the responder popula-tion by polar lipid treated cultured monocytes resultedin significantly reduced proliferative responses to levelscomparable with the unstimulated responder controlcells (Figure 4).

DiscussionMycobacterial lipids have long been implicated in theinteraction between the pathogen and its host. Here wedescribe the consequence of exposure to lipids derivedfrom a virulent M. bovis on the innate immune cells ofcattle.

Figure 4 Proliferative responses (in counts per minute [CPM]) of 1 × 1bars) or polar lipid treated (red bars) MDDC or cultured monocytes. B*** p< 0.001.

Extraction of mycobacterial lipids and their subse-quent analysis by 2D TLC has been previously described[22], but little data exists on total lipid profiling of M.bovis. Previous work by Dandapat et al. [26] attemptedto characterise M. bovis based on the expression of PGLand PDIMs, but only as a tool for identification of theorganism. In Figure 1, we have applied a complete rangeof TLC analyses to polar and apolar lipid extracts whichhas allowed the identification of a broad range of charac-teristic mycobacterial lipids including PDIMS(Figure 1a), the M. bovis characteristic PGL [27] (figure 1B-C), TDM (Figure 1d-e) and PIMs (Figure 1f ). Asexpected, no sulphoglycolipid was found (Figure 1d).Interestingly, TDM was found in both the polar andapolar extracts (Figure 1d-e). This may be related to it’sparticularly amphipathic nature [28] and variable acyl-ation states which are known to alter both immunogen-icity and hydrophobicity [29,30] and may cause themolecule to split differentially across the biphase inter-face during lipid extraction.To discover if the lipid fractions were capable of medi-

ating responses of bovine innate immune cells, stimula-tion experiments were performed and the level of arange of cytokines was analysed. Significant increases inthe production of various cytokines were measured onlyafter cells were exposed to the polar lipid fraction. Per-haps most striking is the significant increase in the pro-duction of the Th-1 polarising IL-12 and the anti-inflammatory cytokine IL-10 (Figure 2a-b). While thismay seem contradictory, it is important to note that thefractions used are complex mixtures of a variety of lipidssome of which are known to induce potent

05 PBMC after allotypic stimulation with either untreated (greyars represent the mean of triplicate wells ± standard error of the mean;


immunostimulatory cytokine profiles, such as MMG[18] and others, such as glycerol monomycolate(GroMM) are known Th2 polarisers [31].Little evidence exists on cytokine production by anti-

gen presenting cells after treatment with lipids, howevermany lipids have been assessed in the context of bothCD4+ and CD8+ T cells. For example, TDM, which ispresent in the polar and apolar fractions (Figure 1d-e),has been shown to induce both Th1 and Th2 cytokines.The induction of IFNγ and IL-12 and the depletion ofIL-4 producing NK cells has been attributed to TDM[32,33] as well as a role, along with IL-6 and TNFα, instable granuloma formation [34]. Yet TDM is also impli-cated in the production of IL-5 and IL-10 in a CD1dependent manner [35]. Furthermore, GroMM has beenimplicated in the induction of Th2 polarising responses[31] where as the closely related GMM has been shownto induce Th1 cytokine responses in T cells [36]. Anti-inflammatory effects have also been attributed to PIM2

and PIM6 where, upon lipid treatment of LPS activatedmacrophages, Doz et al. measured downregulation ofTLR4, TNFα, IL-12p40, IL-6, KC and IL-10 as well asMyD88 mediated NO release [37].Given the significant increase in IL-10 production by

all innate cell types assessed, and the important rolethese cells play in generating and directing the im-mune response, we analysed the expression of antigenpresentation associated cell surface molecules afterlipid exposure. Lipid treatment of APCs leads to a sig-nificant decrease in the levels of costimulatory mole-cules associated with antigen presentation includingMHCII and CD86 on all cell types studied and CD1bon MDDC (Figure 3a-c). Negative regulation of thesemolecules by a variety of lipid components has beennoted previously, especially MHCII in human andmurine systems. Similar to the data presented here, the19-kDa lipoprotein is capable of downregulatingMHCII expression on human THP-1 macrophages byinhibiting activation of the IFNγ - induced CIITA[38,39]. Downregulation of MHCII, as well as TLR2and TLR4, has also been reported on human MDDCafter lipid exposure [40] and a further study also foundimpaired expression of CD1a, MHCII, CD80 and CD83on human MDDC [41].Downregulation of CD1 molecules has also been

shown through the discovery that MDDC generatedfrom BCG treated monocytes did not express CD1 andshowed reduced MHCII, CD40 and CD80 [42] and thishas since been shown to be due to cell wall associatedcarbohydrate α-glucan [43] and mediated through thep38 MAPK pathway [44]. However these experimentshave all been performed in human or murine systemsand with specific lipids, often from avirulent bacterialisolates.

Interestingly, treatment of fresh and cultured mono-cytes with the polar lipid fraction significantly increasedthe level of CD40 expression (Figure 3d) and this effectis not seen on MDDC. This finding seems contradictoryto the published literature [42,45] however these studiesused BCG or TDM alone, rather than the complex andmore biologically representative lipid preparationsderived from virulent mycobacteria used here, as well asbeing performed in human or murine macrophage mod-els. Bovine MDDC expression of CD40 does not alterafter stimulation with either polar or apolar lipids whichmay be due to its constitutively higher levels of expres-sion than on fresh or cultured monocytes.Finally, no difference was seen in the expression of

CD80 after lipid treatment, although this has also beenreported in other systems using virulent M. tuberculosisor avirulent BCG derived lipids [41,42,45].The significant loss of MHCII, CD86 and CD1b is

consistent with the phenotype of an impaired antigenpresenting cell [46]. Given the effect of the polar lipidson the expression of these molecules and the concurrentincrease in IL-10 production, we hypothesised that thepolar lipid fraction, or one of its components, hampersthe ability of the cells to successfully present antigen toT cells and may be able to suppress the induction of aTh1 response during infection. To assess any functionaldeficit in these cells, especially due to the loss in MHCII,lipid treated and untreated cells were used to drive allo-typic proliferative responses.Cultured monocytes drove proliferation of allogeneic

PBMC (Figure 4) and treatment of cultured monocyteswith the polar lipid fraction significantly abrogatedthese responses as suggested by the downregulation ofMHCII and other costimulatory molecules. Proliferativeresponses were also seen when allogeneic PBMC werecombined with untreated MDDC (Figure 4) however nodifference in proliferation was seen using lipid treatedMDDC despite flow cytometric analysis revealing charac-teristic reduction in the level of MHCII on the MDDC(data not shown). While these results seem at odds witheach other, it is possible that the loss of MHCII may beovercome by the high level of CD40 expressed by MDDC(Figure 3d) or the constitutively higher levels of IL-12produced by these cells which further increases signifi-cantly after lipid stimulation (Figure 2b). Also, some evi-dence exists that the presence of CD80 is enough tostimulate allogeneic T cells in the absence of CD86 sig-nalling [47]. Given the significant reduction in CD86 ex-pression on MDDC, the maintenance of CD80 may playa role. Finally, it is possible that, due to constitutivelyhigher levels of MHCII and CD40 present on MDDC, aswell as their expression of CD1b, the levels of MHCIIand CD86 on these cells remains sufficient to drive anallotypic reaction.


These data demonstrate that M. bovis derived lipidfractions are capable of stimulating responses in bovineinnate cells and that these different cell types respond indistinct ways.Interestingly, the alteration in cell surface phenotype

of both cultured monocytes and MDDC seen after polarlipid stimulation is also evident after exposure to theapolar lipid fraction, albeit to a lesser, not statisticallysignificant, extent. This may be due to specific lipidcomponents present in both the polar fraction and theapolar preparation, such as TDM. However, it may alsobe due to the insolubility of less polar lipids in the aque-ous environment an in vitro culture system which maylimit lipid bioavailability.In conclusion, we present here the first data to dem-

onstrate the regulatory effects of M. bovis - derivedlipids on bovine innate cells. These lipids, especiallythose contained within the polar fraction are capable ofinteracting with the host’s innate immune cell’s such thatthe cells ability to initiate an adequate T cell responsemay be compromised, although this effect could only bedemonstrated for cultured monocytes and not MDDC.The lipid fractions used in this study contain the totalfree extractable lipid from M. bovis AF 2122/97, hencewe were not able to attribute these effects to any specificlipid entities. However work is currently being under-taken in our laboratory to further define these responsesand identity the lipids which are responsible for mediat-ing the effects we have shown. Nevertheless, the effectsmediated by these lipids may play a pivotal role in theoutcome of infection and aid further identification of in-dividual lipid components responsible for the immuno-modulatory effects as well as new targets for attenuationand novel vaccine candidates and adjuvant preparations.

Competing interestsThe authors declare that they have no competing interests.

AcknowledgementsThis study was funded by the Department for Environment, Food and RuralAffairs (Defra), UK. The authors would like to express our sincere appreciationto the staff of the Animal Services Unit at AHVLA for their dedication to thewelfare of test animals.

Author details1TB Research Group, Animal Health and Veterinary Laboratories Agency –Weybridge, New Haw, Surrey, Addlestone KT15 3NB, United Kingdom.2School of Biosciences, University of Birmingham, Edgbaston, BirminghamB15 2TT, United Kingdom.

Authors’ contributionsCP - Carried out the studies and prepared the manuscript. SS - Assisted withdendritic cell culture methods. GJ - Participated in the study design andproofing of the manuscript. GB - conceived of the study and participated inits design and coordination. MV - conceived of the study and participated inits design and coordination and helped to draft the manuscript. All authorsread and approved the final manuscript.

Received: 19 March 2012 Accepted: 27 June 2012Published: 27 June 2012

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doi:10.1186/1297-9716-43-54Cite this article as: Pirson et al.: Differential effects of Mycobacteriumbovis - derived polar and apolar lipid fractions on bovine innateimmune cells. Veterinary Research 2012 43:54.

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Highly Purified Mycobacterial Phosphatidylinositol Mannosides DriveCell-Mediated Responses and Activate NKT Cells in Cattle

Chris Pirson,a Regina Engel,b Gareth J. Jones,a Thomas Holder,a Otto Holst,b H. Martin Vordermeiera

TB Research Group, Animal Health and Veterinary Laboratories Agency, New Haw, Addlestone, Surrey, United Kingdoma; Division of Structural Biochemistry, ResearchCentre Borstel, Leibniz-Centre for Medicine and Biosciences, Borstel, Germanyb

Mycobacterial lipids play an important role in the modulation of the immune response upon contact with the host. Using novelmethods, we have isolated highly purified phosphatidylinositol mannoside (PIM) molecules (phosphatidylinositol dimannoside[PIM2], acylphosphatidylinositol dimannoside [AcPIM2], diacyl-phosphatidylinositol dimannoside [Ac2PIM2], acylphosphati-dylinositol hexamannoside [AcPIM6], and diacylphosphatidylinositol hexamannoside [Ac2PIM6]) from virulent Mycobacteriumtuberculosis to assess their potential to stimulate peripheral blood mononuclear cell (PBMC) responses in Mycobacterium bovis-infected cattle. Of these molecules, one (AcPIM6) induced significant levels of gamma interferon (IFN-�) in bovine PBMCs.Three PIM molecules (AcPIM6, Ac2PIM2, and Ac2PIM6) were shown to drive significant proliferation in bovine PBMCs. AcPIM6

was subsequently used to phenotype the proliferating cells by flow cytometry. This analysis demonstrated that AcPIM6 was pre-dominantly recognized by CD3� CD335� NKT cells. In conclusion, we have identified PIM lipid molecules that interact withbovine lymphocyte populations, and these lipids may be useful as future subunit vaccines or diagnostic reagents. Further, thesedata demonstrate, for the first time, lipid-specific NKT activation in cattle.

Members of the mycobacterial genus are renowned for theirwaxy, lipid-rich outer envelope. Under physiological condi-

tions, this outer layer is likely to be the first point of contact be-tween the bacterial cell and the host’s immune system, and theoutcome of this interaction is pivotal in the establishment of in-fection. One of the most important groups of membrane boundlipids consists of the phosphatidylinositol mannosides (PIMs).Interest in PIMs was stimulated since it was shown that phospha-tidylinositol dimannoside (PIM2) forms the phosphoglycolipidanchor which tethers a large array of glycolipids and lipoglycans,including lipomannan (LM) and lipoarabinomannan (LAM), tothe cellular membrane (1). PIMs have been shown to interact witha variety of immune components and mediate significant effectson the host. Ever since the realization that NKT cells could re-spond to lipid antigens (2–4) and the subsequent discovery of theCD1d-restricted lipid antigen �-galactosylceramide (�-GalCer)(5, 6), much research effort has been concentrated on understand-ing lipid antigens. Although work was initially focused on invari-ant NKT cells, it has since been shown that great diversity exists inthe lipid-responsive T cell receptor (TCR) repertoire (7–9) andthat these diverse NKT cells contribute to the Th1/Th2 balance (7,10). It has been shown that CD1d-restricted NKT cells are capableof recognizing a variety of lipid antigens, including phospholipids(11). More recently, a CD1b-restricted subset of T cells has beenfound (12). Similarly to CD1d-restricted invariant NKT (iNKT)cells, the CD1b-restricted variant cells require CD1B for their de-velopment and produce proinflammatory cytokines in responseto CD1b-expressing dendritic cells (DCs) (12). It is clear thatNKT-like cells have a significant role to play in lipid-mediatedresponses, and the hunt for their antigens has continued (13–15).

Given the ability of lipid molecules to generate responses inperipheral blood mononuclear cells (PBMC), we decided to assessthe ability of individual, highly purified natural PIMs to activatebovine lymphocytes. In this study, we developed a novel extrac-tion method which allowed us to extract and highly purify a vari-ety of PIM molecules from virulent Mycobacterium tuberculosis

H37Rv. The ability of these molecules to induce lymphocyte re-sponses in Mycobacterium bovis-infected cattle was investigated bymeasuring lymphocyte proliferation and gamma interferon(IFN-�) production. Furthermore, flow cytometry techniqueswere utilized to characterize responding cell populations.

MATERIALS AND METHODSExtraction of PIMs. Using the novel methodology outlined below,highly pure phosphatidylinositol dimannoside (PIM2), acylphosphati-dylinositol dimannoside (AcPIM2), diacyl-phosphatidylinositol diman-noside (Ac2PIM2), acylphosphatidylinositol hexamannoside (AcPIM6),and diacylphosphatidylinositol hexamannoside (Ac2PIM6) were success-fully isolated. Individual PIM molecules were analyzed by electrosprayionization mass spectrometry (ESI-MS) to confirm identity and purity asshown in Fig. 1. Up to 1 g of dry bacterial mass of Mycobacterium tuber-culosis H37Rv was suspended in 20 to 30 ml of H2O and ruptured utilizinga French press at a minimum of 20,000 kPa. This procedure was per-formed five times, and the combined sample was lyophilized.

Up to 0.5 g of this lyophilized material was extracted three times ac-cording to the method of Bligh and Dyer (16). The dry mass was sus-pended in 4 ml of H2O and washed twice in an additional 2 ml of H2O

Received 1 October 2014 Returned for modification 23 October 2014Accepted 25 November 2014

Accepted manuscript posted online 10 December 2014

Citation Pirson C, Engel R, Jones GJ, Holder T, Holst O, Vordermeier HM. 2015.Highly purified mycobacterial phosphatidylinositol mannosides drive cell-mediated responses and activate NKT cells in cattle. Clin Vaccine Immunol22:178 –184. doi:10.1128/CVI.00638-14.

Editor: D. L. Burns

Address correspondence to Chris Pirson, [email protected].

C.P. and R.E. are joint first authors and contributed equally to this article.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/CVI.00638-14.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/CVI.00638-14

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before being transferred into a 100-ml Erlenmeyer flask. To this samplewas added 30 ml of CHCl3·CH3OH (1:2, vol/vol), and the sample wassonicated (Branson Sonifier 250; output 2, 40% duty cycle, 20 min). Then,10 ml of CHCl3 was added, and the sample was sonicated for a further 5min. Subsequently, an additional 10 ml of H2O was added, and the samplewas sonicated for a final 5 min. The sample was decanted evenly intobetween two and four 50-ml Nalgene Teflon tubes and centrifuged for 30min at 10,000 � g to generate three phases (a water phase, a CHCl3 phase,and an interphase). The water phases (containing LAM and LM) wereremoved, combined, and lyophilized, while the CHCl3 phases were trans-ferred into a single 100-ml pear-shaped flask. The total yield of materialafter three extractions was about 11% of the bacterial dry mass.

Since the remaining interphases contained a lot of AcPIM6, they weresuspended in 8 ml of H2O and combined into 30-ml Kimble high-speedglass tubes before being lyophilized. These phases were then extractedovernight with 30 ml of CHCl3·MeOH (1:2, vol/vol) using a shaker. Thesample was then centrifuged for 30 min at 10,000 � g, and theCHCl3·MeOH phase was removed. This phase was dried under an N2 gasstream before being resuspended in equal parts of CHCl3·MeOH, filteredthrough a 0.2-�m-pore-size polytetrafluoroethylene (PTFE) filter, anddried under N2. This extraction was repeated two times until no PIMscould be identified (total yield of about 2% of the bacterial dry mass). Theobtained PIMs were further purified with silica gel 60 chromatography(see below), as were the CHCl3 phases of the Bligh and Dyer extraction.

Purification of PIMs. All samples were separated on a column (7 by 1cm) of silica gel 60 (0.04 to 0.063 mm) which was successively eluted with(i) 80 ml of CHCl3·MeOH (8:2, vol/vol), (ii) 60 ml of CHCl3·MeOH (1:1,vol/vol), and finally (iii) 40 ml of CHCl3·MeOH·H2O (10:10:3, vol/vol/vol). Coextracted cardiolipin and other lipids eluted in the first mobilephase, most PIMs eluted in the second, and the rest of the PIMs eluted in

the third. Fractions ii and iii were dried under N2, resuspended in 10 ml ofCHCl3·MeOH (8:2, vol/vol), and passed through a 0.2-�m-pore-sizePTFE filter before being analyzed by high-performance thin-layer chro-matography (HPTLC). HPTLC was performed using glass-backed 10- by10-cm silica gel 60 plates (Merck KGAA, Darmstadt, Germany) run inCHCl3·MeOH·H2O (10:8:2, vol/vol/vol) and stained with Hanessian’sstain (0.5 g of ceric sulfate and 25 g of ammonium molybdate in 470 ml ofwater supplemented with 30 ml of sulfuric acid with stirring) and visual-ized at 150°C.

Fractions ii and iii from both of the CHCl3 phases of the Bligh andDyer extraction and the PIMs extracted from the interphase were thenfurther separated by high-performance liquid chromatography (HPLC)using 5-�m Kromasil 100 C18 columns (250 by 20 mm) eluted with eluentA (CHCl3·MeOH·H2O [240:1,140:620, vol/vol/vol] containing 10 mMNH4CH3CO2) and eluent B (CHCl3·MeOH [1,400:600, vol/vol] contain-ing 50 mM NH4CH3CO2). The initial eluent B gradient was 15% for 60min, followed by 20% for 140 min, 40% for 80 min, and finally 100% for60 min at 4 ml min�1. Samples were detected by a light-scattering detector(Sedex; nitrogen pressure, 2 � 105 Pa; temperature, 50°C; split, 1:70).Samples were applied in 200 �l of CHCl3·MeOH (8:2, vol/vol). For ana-lytical runs, 10 �g of sample was injected while 10 mg was injected forpreparative separations.

Since PIM2 and AcPIM6 coeluted on the C18 column, they were sepa-rated by HPLC on 5-�m Prontosil 200-5-C30 reverse-phase columns (250by 4.6 mm) using the same elution reagents. The initial eluent B gradientwas 5% for 5 min, followed by 10% for 15 min, 15% for 50 min, and finally100% for 10 min at 0.8 ml min�1. Samples were injected as a mixture of0.6 mg in 80 �l of CHCl3·CH3OH·H2O (10:10:3, vol/vol/vol) and detectedby the light-scattering detector described above.

FIG 1 Mass spectrometric proof of purity of isolated PIMs. Identified molecular masses of PIM2 (calculated molecular mass, 1176.6784 u) (A), AcPIM2

(calculated molecular mass, 1442.9394 u) (B), Ac2PIM2 (calculated molecular mass, 1653.1378 u) (C), AcPIM6 (calculated molecular mass, 20147.0881 u) (D),and Ac2PIM6 (calculated molecular mass, 2301.3491 u) (E). Letters in parentheses identify the peaks.

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Cattle. Blood samples were obtained from 10 naturally infected, singleintradermal comparative cervical tuberculin test-positive reactors (be-tween 6 and 36 months of age). Animals were sourced from herds withconfirmed bovine tuberculosis breakdowns in Devon, Herefordshire, orWorcestershire and were housed at the Animal Health and VeterinaryLaboratories Agency (AHVLA) at the time of blood sampling. Infectionwas confirmed by necropsy and M. bovis culture in all animals. All proce-dures involving animals were carried out under a project license grantedby the Home Office of Great Britain under the Animals (Scientific Proce-dures) Act 1986. This project was approved by the local VLA AnimalEthics Committee prior to submission to the Home Office.

Isolation of bovine PBMC from whole blood. Whole blood wasmixed in equal parts with sterile Hanks balanced salt solution (HBSS)containing 10 U ml�1 heparin. This mixture was overlaid onto His-topaque 1077 (Sigma-Aldrich) and centrifuged at 800 � g for 40 min. ThePBMC interface was removed using a pastette and washed twice in HBSScontaining heparin. Live cells were identified via trypan blue exclusionand enumerated using a hemocytometer.

Preparation of lipid antigen suspensions. Briefly, lipids were sus-pended in an aqueous phase for use in cell culture experiments after re-moval of CHCl3·CH3OH by evaporation using an N2 gas stream. Cellculture medium was added to the dried lipid, and the mixture was sub-jected to two cycles of heating at 80°C and then sonication for 5 min.Lipids were used to stimulate cells in vitro at 20 �g ml�1 in all assays.

Lymphocyte proliferation assay. Bovine PBMC were prepared fromall 10 animals as described above and were cultured in complete cell cul-ture medium (RPMI 1640 medium containing 25 mM HEPES, 10% fetalcalf serum [FCS], 1% nonessential amino acids [NEAA], 5 � 10�5 mM�2-mercaptoethanol, 100 U ml�1 penicillin, and 100 �g ml�1 streptomy-cin [Gibco Life Technologies, Paisley, United Kingdom]) at 37°C in 5%CO2 for 5 days in the presence of antigen at 2 � 105 cells well�1. After 5days, cells were pulsed with 1 �Ci well�1 of [3H]thymidine overnight,before being harvested using a Harvester 96 Mach III (TomTec, Inc.,Hamden, CT, USA). Lymphocyte proliferation was assessed by the in-creased cellular incorporation of [3H]thymidine (cpm), which was mea-sured using a MicroBeta2 2450 plate counter (PerkinElmer, Waltham,MA, USA). Responses to individual PIMs were considered positive if thecpm exceeded the mean plus 2 times standard deviation of cpm for non-antigen-stimulated cultures from all 10 animals.

Measurement of IFN-� by Bovigam ELISA. Levels of IFN-� in 5-daysupernatants from the proliferation assay were determined using a Bo-vigam enzyme-linked immunosorbent assay (ELISA) kit (Prionics AG,Switzerland). Responses to individual PIMs were considered positive ifthe optical density at 450 nm (OD450) exceeded the mean plus 2 times thestandard deviation of the OD450 for non-antigen-stimulated culturesfrom all 10 animals.

Measurement of proliferation and phenotyping by flow cytometry.Bovine PBMC were isolated as described above and labeled with CellTraceviolet (Invitrogen Molecular Probes, Paisley, United Kingdom) in accor-dance with the manufacturer’s instructions. Briefly, PBMC were sus-pended at 1 � 107 cells ml�1 in prewarmed phosphate-buffered saline(PBS), and 5 mM CellTrace violet was added to a final working concen-tration of 1 �M. Cells were incubated at 37°C for 20 min before unbounddye was quenched with five times the labeling volume of complete cellculture medium at 37°C for 5 min. Finally, cells were pelleted and washedin prewarmed complete cell culture medium, plated at 2 � 105 cellswell�1, and incubated at 37°C in 5% CO2 for 5 days in the presence ofantigen.

Cultured cells were harvested and resuspended in flow cytometry buf-fer (PBS containing 2% FCS and 0.05% NaN3) and labeled for 15 min withthe Near-IR live/dead indicator NIRViD (Invitrogen Life Technologies,Paisley, United Kingdom) and mouse anti-bovine CD335, also known asNKp46 (AKS1; AbD Serotec, Oxfordshire, United Kingdom). Cells werewashed in flow cytometry buffer, and secondary labeling of anti-CD335was performed using a 1:400 dilution of rat anti-mouse IgG2a conjugated

to allophycocyanin for a further 15 min. After a subsequent wash, cellswere further labeled with combinations of R-phycoerythrin (R-PE)-Ze-non-labeled (Invitrogen Life Technologies, Paisley, United Kingdom)mouse anti-bovine CD3 (MM1A; WSU Monoclonal Antibody Centre,Pullman, Washington, USA), mouse anti-bovine CD4 conjugated to Al-exa Fluor 647 (CC30; AbD Serotec, Oxfordshire, United Kingdom),mouse anti-bovine CD8 conjugated to Alexa Fluor 647 (CC63; AbD Se-rotec, Oxfordshire, United Kingdom), and Alexa Fluor 488-Zenon-la-beled (Invitrogen Life Technologies, Paisley, United Kingdom) mouseanti-bovine ��-TCR1 (GB21a; WSU Monoclonal Antibody Centre, Pull-man, Washington, USA). Finally, labeled cells were washed in flow cytom-etry buffer and resuspended in 150 �l of 2% paraformaldehyde (Cytofix;BD Biosciences, Oxfordshire, United Kingdom) for at least 30 min at 4°Cbefore analysis on a CyAn ADP analyzer. For capture and analysis, initialgating was on single, NIRViDlo (live) cells into a subsequent lymphocytegate before gating on CellTrace violetlo cells.

Data and statistical analysis. All data representation and statisticalanalysis were performed using GraphPad Prism, version 5.04, and Graph-Pad InStat, version 3.06 (GraphPad Software, La Jolla, CA, USA). Statis-tical analysis of IFN-� and lymphocyte proliferation data was performedusing a nonparametric repeated-measures analysis of variance (ANOVA;Friedman test) with a Dunn’s multiple comparisons posttest.

RESULTSHighly purified PIM molecules can be isolated from virulentmycobacteria. PIM molecules differing in the number of acyl andmannose residues were highly purified from M. tuberculosis. Fivedistinct PIM molecules were isolated: PIM2, AcPIM2, Ac2PIM2,AcPIM6, and Ac2PIM6. In total, these lipids constituted 3% of thebacterial dry mass after silica gel 60 separation. The ability of thepurification method to isolate highly pure PIMs is shown in Fig. 1,in which the structures and purity of the different PIM moleculeswere confirmed by ESI-MS. In addition, bands corresponding toAcPIM2 molecules (that differed only in the number of carbonatoms in the acyl chains) were clearly resolved by thin-layer chro-matography (TLC) analysis (see Fig. S1 in the supplemental ma-terial).

Purified PIM molecules activate lymphocytes from M. bovis-infected cattle. In order to assess the ability of purified PIMs toinduce in vitro immune responses in cattle, PBMC from 10 natu-rally M. bovis-infected cattle were cultured for 5 days in the pres-ence of each PIM molecule, and the level of IFN-� was measuredby ELISA (Fig. 2A). Both the frequency and strength of IFN-�responses differed depending on the nature of the PIM molecule.AcPIM2 was least recognized, inducing responses in only 2 of the10 animals. PIM2, Ac2PIM2, and Ac2PIM6 were more frequentlyrecognized, with responses detected in 3 (PIM2) and 4 (Ac2PIM2

and Ac2PIM6) out of 10 animals. AcPIM6 was most frequentlyrecognized, inducing responses in half of the animals studied. Fur-thermore, AcPIM6 was the only PIM molecule to induce signifi-cantly greater levels (P 0.01) of IFN-� overall than nonstimu-lated controls.

In addition to measuring IFN-� production, we also investi-gated the ability of the PIM molecules to induce PBMC prolifer-ative responses in the same animals. Again, the frequency of re-sponding animals differed depending upon the nature of the PIMmolecule (Fig. 2B). PIM2 failed to induce a proliferative responsein any of the animals studied, while AcPIM2 induced responses inonly 3 out of 10 animals. In contrast, Ac2PIM2, AcPIM6, andAc2PIM6 were more frequently recognized, inducing proliferativeresponses in 6 (Ac2PIM2 and AcPIM6) and 7 (Ac2PIM6) out of 10animals. Overall, significantly greater PBMC proliferation was de-

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tected in the Ac2PIM2, AcPIM6, and Ac2PIM6 treatment groupsthan in nonstimulated controls, with median values tending to begreater following AcPIM6 stimulation.

Phenotyping of AcPIM6-responsive proliferating cells byflow cytometry. As AcPIM6 was the only PIM molecule to gener-ate significantly increased levels of IFN-� (Fig. 2A) and producedthe greatest increase in the median proliferative response (Fig.2B), we used this antigen to stimulate PBMC from an M. bovis-infected animal to characterize the proliferating cell populationsby flow cytometry. Purified protein derivative from M. bovis(PPD-B) was used as a control antigen. CellTrace violet-labeledcells were incubated for 5 days with antigen before being harvestedand labeled for flow cytometric analysis. After stimulation witheither PPD-B or AcPIM6, three populations of proliferating cellswere identified based on cell surface phenotyping: (i) CD4 T cells(CD3 CD4), (ii) CD8 T cells (CD3 CD8), and (iii) NKT-like cells (CD3 CD335). An example of the gating strategy foridentifying proliferating NKT cells is highlighted in Fig. 3A, whichdemonstrates a greater level of proliferating cells in response tostimulation with AcPIM6 (58.29%) than in the non-antigen-stim-ulated control (29.87%).

The effects of stimulation with either PPD-B or AcPIM6 on thethree different cell populations are summarized in Fig. 3B. Stim-ulation with PPD-B drove antigen-specific proliferation of ap-proximately 60% of the CD4 T cells (CD3 CD4). Similarly, anantigen-specific proliferative response was seen in approximately15% of the CD8 T cells (CD3 CD8) to PPD-B. A slight in-crease in NKT cell (CD3 CD335) proliferative responses (ap-proximately 20%) was also seen to these antigens (Fig. 3B).

Stimulation with AcPIM6 induced only limited proliferation ofCD4 T cells (approximately 5%) and no proliferation of CD8 Tcells above the background (Fig. 3B). In contrast, approximately30% of the NKT cell population mounted a proliferative responseafter stimulation with AcPIM6 (Fig. 3B). Little or no proliferationabove the unstimulated negative control was seen in the CD3�

CD335 cells or in the CD3 ��-TCR populations (data notshown).

DISCUSSION

Mycobacterial lipids have long been implicated in the induction ofresponses in both the innate and adaptive cell-mediated immune

responses (17–21). Although one strategy has been publishedwhich allows the isolation of PIMs from the avirulent M. bovisBCG strain (18, 22–24) and M. tuberculosis H37Rv (22) and cer-tain synthetic molecules (17, 19, 21), in this study we successfullydeveloped a novel method for extracting and subsequently highlypurifying and characterizing individual PIM species from the po-lar fraction of virulent M. tuberculosis H37Rv. The method devel-oped here improves upon the previously published protocols pri-marily by using a French press to disrupt the bacterial cells,thereby increasing PIM yield. Other refinements include the re-moval of the hot acetone incubation and the use of different re-verse-phase conditions for PIM purification. Our strategy allowedus to isolate a greater yield of more highly purified PIMs, as con-firmed by ESI-MS, that could be subsequently assayed for theirability to generate responses in lymphocytes.

To assess the ability of these highly purified PIMs to drive im-mune responses, the individual molecules were used to stimulateperipheral lymphocytes isolated from M. bovis-infected cattle.Only AcPIM6 drove significant levels of IFN-� from PBMC (Fig.2A). Interestingly, when whole blood taken from the same animalswas stimulated overnight with the PIM molecules as previouslydescribed (25-27), no IFN-� could be measured, and this was notdue to a lack of viability as stimulation with pokeweed mitogen(PWM) generated high levels of IFN-� (data not shown). Al-though no IFN-� production was seen from the whole-blood as-say, AcPIM6 was able to drive significant production of IFN-�from PBMC incubated for 5 days (Fig. 2A).

There are very few studies showing the effect of mycobacteriallipids in short-incubation, whole-blood assays. Cell-mediated im-mune responses to lipid antigens are more commonly assessed byenzyme-linked immunosorbent spot (ELISpot) assay, with incu-bation times of at least 48 h required before measurable responsesbecome apparent (24, 28). Further, the requirement for antigenprocessing and presentation of specific PIMs has been demon-strated previously (20); perhaps the most likely explanation forthe discrepancy between whole-blood and PBMC IFN-� re-sponses is that the frequencies of lipid-responsive cells are low andthat an extended incubation allows for expansion of these cells.This is supported by our demonstration of strong proliferativeresponses induced after stimulation of PBMC with PIMs (Fig. 2B).

FIG 2 (A) PIM-driven IFN-� production as measured by Bovigam ELISA. PBMC were incubated with PIMs at 20 �g ml�1 for 5 days. Points represent meanresponses from triplicate wells for each of the 10 animals tested; lines represent the sample median. ***, P 0.001. (B) PIM-driven proliferation of bovine PBMCas measured by [3H]Thy incorporation. Bovine PBMC were stimulated for 5 days with individual PIMs at 20 �g ml�1. Points represent mean responses fromtriplicate wells for each of the 10 animals tested; lines represent the sample median. *, P 0.05; **, P 0.01. Nil, nonstimulated controls.

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A recent study has shown that bovine NKT cells are present only atlow frequencies (0.1% to 1.7%) (29).

Previous work using mice has shown that the ability of PIMs togenerate cell-mediated responses is dependent on the acyl struc-tures of the molecules. Early work performed using PIM2 andPIM6 demonstrated that the acyl chain was essential for NKT cell

recruitment while the complexity of the mannose residues did notalter the response (23); however, it was subsequently shown thatthe second acyl chain of PIM4 enhances binding to murine CD1dbut that the polar mannose head was essential for antigen recog-nition, proliferation, and IFN-� production (28). As well as thenumber and location of acyl chains, their degree of unsaturation

FIG 3 Assessment of proliferating cell phenotype by flow cytometry. (A) Flow cytometric gating strategy. Single, live CD3 CD335 lymphocytes were assessedfor CellTrace violet labeling, and cells expressing low levels of CellTrace violet were gated for phenotyping. Numbers represent the percentages of proliferatingcells in response to each antigen. FS Lin, forward scatter (linear). (B) Proliferation of CD3 CD4, CD3 CD8, or CD3 CD335 cells in response to eitherPPD-B or AcPIM6. Each bar represents the percentage of cells proliferating after subtraction of the unstimulated control.

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and cis, but not trans, stereochemistry are critical in determiningantigenicity (14, 30).

The larger and more complex PIM molecules tested here(Ac2PIM2, Ac2PIM6, and AcPIM6) drove significant levels of pro-liferation. AcPIM2 also drove proliferation in 3 animals whilePIM2 generated no positive responses.

As AcPIM6 was the only molecule to drive significant IFN-�responses in our study (Fig. 2A) and one of the most potent in-ducers of proliferation (Fig. 2B), we decided to use AcPIM6 tocharacterize the proliferative response. Stimulation with AcPIM6

induced higher levels of proliferation in NKT cells than in CD4

or CD8 T cells (Fig. 3). However, from these data it is not possi-ble to tell if the proliferative CD4 or CD8 cells also coexpressCD335 as our flow cytometric labeling panels do not allow thediscrimination; however, this is a distinct possibility.

Although well characterized in humans and mice, the presenceof NKT cells in cattle has been a controversial issue (31–34). Nev-ertheless, studies have shown that the bovine CD1D gene is ex-pressed and translated in vivo (35), and recent work has identifieda subset of cattle lymphocytes that express both T cell (CD3) andNK cell (NKp46) markers, suggesting the presence of an NKT cellpopulation in bovine peripheral blood (29). Furthermore, bovineNKT cells have been shown to express both ��- and ��-TCRs, tohave a broad TCR repertoire, and to have fully functional NKp46,CD16, and CD3 signaling pathways (29). Interestingly, these cellsrequire ligation of their CD3 molecules to produce IFN-�. Whilethis may initially suggest that a CD3 binding component may bepresent in our PIM preparations, it is worth noting that we havenot identified the cytokine-producing cells.

The identification of AcPIM6 as a potent immunostimulatorymolecule is of great interest both as a potential vaccine candidate(21), an adjuvant formulation (36), or a target for attenuation inthe development of novel live vaccines (37). PIMs have also beenused previously as diagnostic reagents for both tuberculosis andleprosy, although with limited success (38).

In conclusion, we present here the ability to extract and selec-tively purify PIMs to a high level of purity. These molecules couldbe used to stimulate significant IFN-� production and drive sig-nificant proliferation in PBMC from cattle. We have also been ableto identify the proliferative population and, for the first time, wehave shown antigen-specific NKT activation in cattle.

ACKNOWLEDGMENTS

This study was funded by the Department for Environment, Food andRural Affairs, United Kingdom (EMIDA-funded project Mycobactdiag-nosis).

We sincerely appreciate the staff of the Animal Services Unit atAHVLA for their dedication to the welfare of test animals. We also thankFlorence Dufreneix for her technical assistance and Buko Lindner (Re-search Centre Borstel) for ESI-mass spectrometry.

We have no competing interests.

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Differential effects of Mycobacterium bovis - derived polar and apolar lipid fractions on bovine innate immune cells

Chris Pirson1, Gareth J Jones1, Sabine Steinbach1, Gurdyal S Besra2, H Martin Vordermeier1

1Department for Bovine TB, Animal Health and Veterinary Laboratories Agency, Woodham Lane, New Haw, Addlestone, Surrey, KT15 3NB2 School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT

Figure 1:

A - D: Apolar fraction analysedWith standard TLC systems A, B, C and D

E and F: Polar fraction analysed with stadard TLC systems D and E

PDIM - phthiocerol dimycocerosateMQ - menaquinoneTAG - triacyl glycerolPAT - pentacyl trehalosePGL - phenolic glycolipidMMG - monomycolyl glycerolTMM - trehalose monomycolateTDM - trehalose dimycolateGMM - glucose monomycolateDPG - diphosphatidyl glycerolPE - phosphatidyl ethanolaminePI - phosphatidyl inositolP - phospholipidPIM - phosphatidyl inositolmannosides

Cytokine responses toM. bovis lipidsMonocytes were extracted from bovine whole blood and cultured with GM-CSF and IL-4 (MDDC) or GM-CSF alone (CM) for 3 days before being washed and stimulated overnight with lipid at 20 μg ml-1 .

Supernatants were analysed by MSD multiplex chemiluminescent ELISA for the levels of IL-10, IL-12, MIP-1β, TNFα and IL-6 (figure 2).

Polar, but not apolar, lipids caused significant increases in production of IL-10, IL-12, MIP-1β and TNFα in all cells. DC produced more IL-12 and TNFα than other cells, while DC and CM produced more MIP-1β than monocytes.

Phenotypic responses to M. bovis lipidsAnalysis of antigen presentation associated molecules was performed by flow cytometry (figure 3).The polar lipid fraction caused a significant reduction in expression of MHCII and CD86 on all cell types, as well as significant reduction in CD1b expression on MDDC.CD40 levels increased significantly after polar lipid stimulation of cells where expression was not constitutively high.

Figure 3:Surface expression of MHCII (A), CD86 (B), CD1b (C) and CD40 (D) on cultured cells and fresh monocytes after exposure to lipid fractions. ** p<0.01; *** p<0.001; Repeated Measures ANOVA w. Bonferroni Multiple Comparisons Test

Functional consequence of lipid exposureAn MLR was performed to assess the consequence of reduction in expression of MHCII. Cultured cells were Mitomycin C treated before being mixed with pre-screened alloreactive PBMC from a different animal. Polar lipid treatment of CM

led to statistically significant abrogation of proliferative responses. However, exposure of MDDC to lipids had no effect upon their ability to drive alloreactive proliferation. This may be due to constitutively high levels of CD40 found on MDDC or the significantly higher levels of IL-12 produced by these cells (figure 4).

Figure 4:PBMC proliferative responses after allotypic stimulation with untreated (light grey bars) or polar lipid treated (dark grey bars) cultured cells. Bars represent the mean of triplicate wells ± standard error of the mean. *** p<0.001; One Way ANOVA w. Bonferroni Multiple Comparison Test

ConclusionsWe present here the first data to fully categorise the lipids of M. bovis and demonstrate their regulatory effects of on bovine innate immune cells. Polar lipids induced significant levels of IL-10, IL-12, MIP-1β and TNFα production by innate immune cells. Polar lipids also caused a significant reduction in expression of MHCII, CD86 and CD1b. Finally, polar lipid treated cultured monocytes were shown to be compromised in the induction of T cell proliferation. Hence, polar lipids may play a pivotal role in the outcome of infection.

Acknowledgements & Contact DetailsThis study was funded by the Department for Environment, Food and Rural Affairs (defra), UK.The authors would like to express our sincere appreciation to the staff of the Animal Services Unit at AHVLA for their dedication to the welfare of test animals.

Chris Pirson can be contacted at:• [email protected]• +44 (1932) 357662• www.chrispirson.co.uk

IntroductionThe first point of contact between M. bovis and its host is likely to be an interaction between the innate immune system and surface expressed molecules of the bacilli. The mycobacterial cell surface contains large numbers of diverse and complex lipids which are known to interact with innate cell receptors.Little data exist describing the effect of pathogen derived lipids on innate immune cells from the appropriate host. Thus, to assess the immune response of the host to the lipids of its natural pathogen, lipids were extracted from virulent M. bovis AF 2122/97 and used to stimulate various bovine innate immune cells isolated from live, TB free, cattle.

M. bovis AF 2122/97 derived lipidsLipids were extracted from freeze dried M. bovis AF 2122/97 in 2 fractions; apolarlipids were extracted in petroleum ether and polar lipids in chloroform and methanol. Lipids were analysed using standard 2D TLC systems and individual lipid species were identified (figure 1).

Figure 2:

IL-10 (A), IL-12 (B), MIP-1β (C), TNFα (D) & IL-6 (E) production by monocytes and cultured cells in response to stimulation with lipid fractions.

* 0<0.05; ** p<0.01; *** p<0.001Kruskal-Wallis w. Dunn’s Multiple Comparisons Test

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